Search

The aim of the study was to design a construct based on a nasal septal cartilage plate providing required cell differentiation in different layers to replace a deep osteochondral defect and develop an algorithm of chemical and physical effect sequence to create non-immunogenic two-layer porous structure with requisite elasto-mechanical properties. Materials and Methods The plates derived from porcine nasal septal hyaline cartilage covered by perichondrium were multi-stage treated including freezing, equilibrating in a hypotonic saline solution (type I specimens); trypsinization, point IR-laser effect, re-trypsinization (type II specimens); a stabilizing effect of crosslinking agents — glyceraldehyde/ribose in an acidic medium — washing (type III specimens). For all type specimens: there were established stability parameters (collagen denaturation temperature using a thermal analysis; and Young’s modulus using a mechanical analysis); there were determined morphological characteristics using light and polarization microscopy with classical staining and nonlinear optical microscopy in second-harmonic generation mode. Results Thermal, mechanical, and morphological properties in type I specimens slightly differed from those of the initial nasoseptal system. A considerable part of cells had destroyed membranes. In type II specimens, thermal stability of collagen frame was significantly lower; Young’s modulus decreased more than fourfold compared to type I specimens. Collagen structure of hyaline cartilage appeared to be disarranged, although the morphological differences of the hyaline part and perichondrium preserved. The construct matrix was almost completely decellularized. Successive exposure to laser radiation and trypsin resulted in the formation of partial holes in the matrix, ~100 μm in diameter. In type III specimens, both the thermal stability of the collagen frame and Young’s modulus (E) increased. Glyceraldehyde was more effective than ribose, E having reached the value typical for intact hyaline cartilage. Collagen fibers in type III specimens were thicker than in type I and II specimens. The morphological differences of the hyaline part and perichondrium and partial holes were preserved. Conclusion Due to sequential treatment by salts, trypsin, IR-laser radiation, and nontoxic crosslinking agents, nasal septal cartilage plate forms porous acellular construction consisting of two layers formed by type I (from perichondrium) and type II (from hyaline part) collagen fibers. In the present construction, stability, mechanical properties, and size of the partial holes can be assigned for cell colonization. It enables to use the construction to replace articular cartilage defects.

The aim of the investigation was to study a systemic level of L-DOPA, dopamine, and norepinephrine, and assess their prognostic value in retinopathy of prematurity (ROP) development on an experimental disease model. Materials and Methods The investigation was carried out on infant Wistar rats (n=36) divided into a study group (rat infants with experimental ROP, n=17) and a control group (n=19). The animals of both groups were sacrificed on days 14, 21–23, and on days 28–30. The choice of the indicated periods corresponded to the key stages of ROP development in an experiment and was based on the findings of our previous histological studies. Dopamine, L-DOPA, and norepinephrine levels in infant rat blood plasma samples were determined. Results On day 14 of the experiment (the period corresponds to the pathological neovascularization induction in the applied model and preclinical ROP in children), mean L-DOPA level in infant rats with ROP (0.31 ng/ml) was significantly decreased compared to that in the controls (0.42 ng/ml) (p≤0.01). On days 21–23 of the experiment (the period corresponds to the growth of pathological extraretinal neovascularization in the applied model and ROP stage 3 in children) the systemic level of L-DOPA was still statistically reduced in the study group (0.87 ng/ml) compared to the control group (1.53 ng/ml) (p≤0.01). On days 28–30 of the experiment (the period corresponds to the regress of neovasculature in the applied model and a spontaneous ROP regress stage in children) the L-DOPA level in blood plasma in the study group (0.33 ng/ml) showed an insignificant upward tendency in reference to the controls (0.21 ng/ml). Mean dopamine and norepinephrine levels had no difference in the groups under study of infant rats within all follow-up periods. Conclusion Low systemic level of L-DOPA at the preclinical stage of experimental ROP should be considered as a laboratory prognostic criterion of a developing pathological process; it will enable to use the criterion when working out the measures to optimize the existing screening system for the disease in children.

The aim of the study was to assess the levels of Всl-2 and Bax proteins in the vascular wall and their correlation with serum cholesterol in patients with stage III–IV atherosclerosis obliterans of lower limb arteries. Materials and Methods The study included 32 patients with stage III–IV atherosclerosis obliterans of the lower limb. Samples of intraoperative material (all three layers of the vascular wall) including an atherosclerotic plaque (AP) were taken during primary open surgery on major leg arteries. As a control, we used samples of the arterial wall without visible signs of atherosclerosis. Based on AP ultrasonography, the patients were divided into two groups: with APs of mixed echogenicity and with hyperechoic (calcified) AP. The vascular samples were crushed and homogenized for further measurements of Всl-2 and Bax proteins; in a separate setup, cholesterol in blood serum was measured. Results In patients without atherosclerotic changes, the level of the anti-apoptotic protein Bcl-2 in the arterial wall was 1.25 ng/mg, and that of the pro-apoptotic protein Bax — 4.7 ng/mg. In the case of APs of mixed echogenicity, the expression of Bcl-2 was 1.8 ng/mg (p=0.143) and that of Bax — 5.1 ng/mg (p=0.834), with no significant differences from AP-free vascular wall samples. In the arterial wall containing a heterogeneous calcified AP, the expression of Bcl-2 was 0.9 ng/mg (p=0.143), In contrast, the level of Bax was 6.8 ng/mg, which showed its significant increase as compared with the non-AP controls (p=0.02). In the cases with predominantly hyperechoic AP, the expression of Bcl-2 was significantly lower (p=0.036), and that of Bax — significantly higher (p=0.036) in comparison with AP of mixed echogenicity. In patients with hyperechoic AP, we found a negative correlation between the Bax and Bcl-2 values ( r=–0.315) and a positive correlation between the Bax expression and serum cholesterol (r=0.617). Conclusion In arterial walls with hyperechoic (calcified) APs, the expression of anti-apoptotic protein Bcl-2 is reduced, and that of pro-apoptotic protein Bax is increased, which indicates the apoptosis activation in advanced atherosclerotic lesions. In patients with such APs, elevated cholesterol levels directly correlate with the increased expression of pro-apoptotic Bax protein (r=0.617).

The aim of the study was to explore the association between sudden cardiac death (SCD) and single nucleotide polymorphisms (SNPs) rs34554140, rs6670279, and rs6874185 from the list of potential molecular genetic markers of SCD, obtained in our earlier genome-wide allelotyping on pooled DNA samples. Materials and Methods The study is based on the case-control principle. The SCD group included 438 deceased residents of Novosibirsk (average age — 53.2±9.1 years; men — 72.7%, women — 28.3%) with the main postmortem diagnoses of acute circulatory failure or acute coronary failure, which met the criteria of SCD established by the European Society of Cardiology. The control group included 435 live subjects enrolled in the international projects HAPIEE and MONICA (average age — 53.2±8.9 years; men — 70.0%, women — 30.0%). DNA was isolated by phenol-chloroform extraction from the myocardial tissue in the SCD group and from the venous blood in the control group. Genotyping was performed by polymerase chain reaction with subsequent analysis of restriction fragment length polymorphism in a polyacrylamide gel. Results The frequencies of the genotypes of SNPs rs34554140, rs6670279, and rs6874185 in the control group correspond to those predicted by the Hardy–Weinberg equilibrium (c2=0.98, 0.009, 3.39, respectively). The AA genotype of rs34554140 is associated with an increased risk of SCD (p=0.002; OR=1.85; 95% CI 1.26–2.71). The AT genotype has a protective effect against SCD (p=0.001; OR=0.53; 95% CI 0.36–0.78). In subgroups separated by gender and age, the differences persist in the subgroups of men, women, and individuals under 50 years old (p0.05). Conclusion The association of single nucleotide polymorphisms rs34554140 and rs6670279 with SCD was confirmed. In contrast, no association of rs6874185 with SCD was detected.

The aim of the investigation was to study the influence of pore size and the presence of a biologically active calcium phosphate coating in porous 3D printed titanium implants on the process of integration with the bone tissue. Materials and Methods Samples of cylindrical implants with three different pore diameters (100, 200, and 400 μm) were fabricated from titanium powder on the Arcam 3D printer (Sweden) using electron beam melting technology. A calcium phosphate coating with a thickness of 20±4 μm was applied to some of the products by microarc oxidation. Cytotoxicity of the implants was determined in vitro on human dermal fibroblast cultures. The samples were implanted in the femoral bones of 36 rabbits in vivo. The animals were divided into 6 groups according to the bone implant samples. The prepared samples and peri-implant tissues were studied on days 90 and 180 after implantation using scanning electron microscopy and histological methods. Results All samples under study were found to be non-toxic and well biocompatible with the bone tissue. There were revealed no differences between coated and non-coated implants of 100 and 200 μm pore diameters in terms of their histological structure, intensity of vascularization in the early stages, and bone formation in the later stages. Samples with pore diameters of 100 and 200 μm were easily removed from the bone tissue, the depth of bone growth into the pores of the implant was lower than in the samples with pore diameter of 400 μm (p

Platelet-derived growth factor (PDGF) plays an important role in angiogenesis, affects activation of migration and proliferation of mesenchymal stem cells, fibroblasts, smooth muscle cells, osteoblasts; activation of migration of monocytes, macrophages, and neutrophils. The aim of the investigation was to study the effect of cryo-processing on the qualitative properties of platelet-rich autoplasma (PRP) at different time intervals. Materials and Methods. Autologous plasma preparations were obtained from the blood of 31 donors. The biological material was prepared by double centrifugation according to the protocol for obtaining P-PRP and L-PRP. Platelet count and the concentration of growth factors (PDGF-AA and PDGF-BB) were studied in fresh PRP preparations. In frozen PRP samples, the concentration of PDGF-AA and PDGF-BB was determined 2 weeks after cryo-processing and 2 months after cryo-processing at –35 °С. P-PRP and L-PRP samples activated with 10% CaCl2 solution and those non-activated were studied. Results. L-PRP preparations are significantly superior to P-PRP preparations: the concentration of platelets is 1.7 times higher in them. The level of PDGF-AA in non-activated L-PRP is 1.8 times higher than in non-activated P-PRP (p0.05). Conclusion. Cryo-processing of non-activated autologous L-PRP allows preserving and subsequently enhancing the properties of plasma concentrate, which makes it possible to apply it in clinical practice.

The aim of the investigation was to study the biodegradation characteristics and rate of magnesium alloys in vitro. Materials and Methods. We studied the biodegradation of magnesium alloys Mg-Zn-Ca and WE43 (Mg-Y-Nd-Zr) in homogenized (initial) condition and after strengthening by mechanical processing using equal channel angular pressing (ECAP). The samples were incubated in a model system based on reference fetal calf serum (FCS) in the static and dynamic modes. The morphology of alloy surfaces was analyzed using light microscopy and computed tomography. Biodegradation was assessed by calculating weight loss within a certain incubation period. Cell adhesion and colonization stimulation were quantified in terms of a cell index (CI) using an analyzer xCELLigence RTCA Systems (ACEA Biosciences, Inc., USA) during the incubation of HEK 293 cells on WE43 specimens. Results. Strengthening of magnesium alloys Mg-Zn-Ca and WE43 using ECAP and, consequently, the changed structure resulted in the biodegradation acceleration as high as eightfold. Among the specimens incubated in FCS in different modes, those incubated in liquid flow exhibited the biodegradation rate twice as high as that of the specimens tested under static conditions. The biodegradation process was accompanied by local corrosion, although the degradation was primarily concentrated along the specimen margins stimulating cell adhesion and colonization. Such nature of degradation, as a rule, does not lead to anisotropy of the strength characteristics, that is important for medical materials. Superficial degradation of the alloys with no X-ray density changes in the bulk of the specimens was confirmed by computed tomography. Conclusion. The study of the biodegradation rate and further characteristics of magnesium alloys Mg-Zn-Ca and WE43 showed that the materials in both structural conditions are suitable for implants and can be used in bone implants and surgical fasteners.

We previously found that the number of CCR6+ T-helpers with the phenotype of effector/effector memory T cells increases in the blood of patients with H. pylori-associated peptic ulcer. The mature phenotype and the expression of the chemokine receptor CCR6, which is involved in migration of lymphocytes to the inflamed mucous membrane of the gastrointestinal tract, suggests that these cells are involved in the immune response observed in this clinical condition. To better understand the pathogenetic role of these cells, it is necessary to study their functional activity, specifically, the production of pro-inflammatory cytokines involved in the pathogenesis of the disease. The aim of the study was to evaluate changes in the blood level of pro-inflammatory types of mature CCR6+ T-helpers in H. pylori-associated peptic ulcer disease. Materials and methods CCR6+ T-helpers were isolated from the blood by using immuno-magnetic separation adapted to this study. The number of T-helpers of types 1 and 17 (Th1 and Th17) and cells with mixed properties of Th1 and Th17 (Th1/Th17) was determined by intracellular cytokine assay. Results Initially, we planned to activate unseparated peripheral blood mononuclear cells ex vivo and evaluate the number of cytokine producers among mature CCR6+ T-helper cells by gating them during the flow cytometry. However, dramatic changes in the phenotype of T-helpers upon activation did not allow us to reliably identify the cells of interest. Subsequently, we used a two-stage immunomagnetic separation procedure to obtain functionally active mature CCR6+ T-helpers with a purity of >90%. The quantitative yield of these cells from the blood of patients with gastric and duodenal peptic ulcer associated with H. pylori was 9 times higher than that from the blood of healthy donors. Activation of CCR6+ T-helpers purified from blood of ulcer patients revealed an increased content of Th1, Th17, and Th1/Th17. One ml of the patient's blood yielded 18.1 times more CCR6+ Th1, 19.4 times more CCR6+ Th17, and 21.1 times more CCR6+ Th1/Th17 compared with the blood of healthy subjects. Conclusion The content of mature CCR6+ T-helper cells with pro-inflammatory activity significantly increases in the blood of patients with peptic ulcer associated with H. pylori infection.

Although major progress has been made in the standard treatment for glioblastomas, encompassing the maximal surgical resection, chemotherapy and radiation therapy, it is possible to increase survival rates significantly only in a few patients. Therefore, it is necessary to explore new therapeutic modalities, one of which is immunotherapy. The aim of the study was to evaluate the efficacy of the combined use of autologous and pooled tumor lysates in comprehensive treatment of patients with glioblastoma. Materials and Methods All patients (n=58, including 30 males and 28 females aged 18–70 years) were randomized into three groups, two of which received immunotherapy based on injection of autologous dendritic cells pulsed with autologous tumor lysates (first protocol) or pooled lysates (second protocol) from more than one tumor, in addition to the planned standard treatment. The patients of group 3 (control) received the standard comprehensive treatment encompassing the maximum safe tumor resection followed by radiation therapy and chemotherapy. Results The tolerability of both applied immunotherapy protocols was good: there were no anaphylactic reactions observed or patients who prematurely discontinued participation in the study. The final analysis of the data revealed no significant differences in median survival values of patients in each of the three groups. However, when analyzing the Karnofsky Performance Status in patients of group 2, it was found that it tended to improve. Conclusion The study shows that the proposed immunotherapy protocols are safe for clinical use and have the potential to improve the patient’s life quality. However, these findings should be considered intermediate until the findings of multicenter randomized clinical trials with a larger number of patients are obtained.

The aim of the study was to assess the possibility of using plasma levels of tumor M2-pyruvate kinase (Tu M2-PK), matrix carbonic anhydrase IX (CA9), and matrix metalloproteinase 9 (MMP9) in patients with renal cell cancer as predictors of the disease course and the response to treatment. Materials and Methods Samples of blood plasma or serum of 46 patients with clear cell renal cancer T1–4N0–1M0–1 obtained before surgery and 8–9 days after surgery were tested. The control group consisted of 20 practically healthy individuals, comparable in age with the examined patients. Quantitative determination of Tu M2-PK in EDTA-added blood plasma was performed by enzyme-linked immunosorbent assay using a ScheBo Tumor M2-PK test (Germany). Determination of CA9 by ELISA was performed using a Human Carbonic Anhydrase IX Quantikine ELISA Kit (USA) and MMP9 — using a Quantikine ELISA Kit (USA). Results In patients with renal cell carcinoma, a statistically significant increase in the level of Tu M2-PK, CA9 and a statistically significant decrease in MMP9 in comparison with the control group were found. The level of Tu M2-PK in patients with localized kidney cancer was significantly lower than in patients with disseminated cancer. An increase in size of the primary tumor and a decrease in the degree of its differentiation correlated with an increase in Tu M2-PK, and decrease in CA9 and MMP9 in the blood serum. Performing surgery equivalent to nephrectomy did not change the Tu M2-PK levels in the early postoperative period, but caused a decrease in the levels of CA9 and MMP9. Conclusion The results indicate a potential significance of Tu M2-PK, CA9, and MMP9 as biological markers for predicting the disease course in patients with renal cell carcinoma.

The aim of the investigation was to study the integration between native human dental tissue and new-generation biomimetic materials replicating the mineral-organic complex of dentin and enamel using IR microspectroscopy for multidimensional visualization and analysis. Materials and Methods The conditions for stable integration at the interface between biomimetic material and natural hard tissue were identified using a biocomposite buffer system of nanocrystalline carbonate-substituted calcium hydroxyapatite corresponding in its total characteristics to human dentin-enamel apatite and a number of amino acids present in the organic matrix of dentin and enamel: L-histidine, L-lysine hydrochloride, L-arginine hydrochloride, and hyaluronic acid. The finished samples were studied using IR microspectroscopy on IRM channel equipment (The Australian Synchrotron, Melbourne, Australia). Results The characteristic features of the biomimetic buffer layer at the interface between the enamel and dental material were revealed and visualized based on IR mapping of absorption intensity for particular functional molecular groups with the use of synchrotron radiation, location of the functional groups involved in the processes of biomimetic composite integration was identified.

The aim of the study was to define the role of brain-derived and glial cell line-derived neurotrophic factors (BDNF and GDNF) in realization of compensative and adaptive mechanisms of a neonatal organism to hypoxia. Materials and Methods The experiments in vivo have been carried out on pregnant C57BL/6 mice (n=36). Chronic hypobaric hypoxia has been modeled in different pregnancy trimesters. On gestation days E19–20, concentration of BDNF and GDNF in the blood of the pregnant females was determined by enzyme immunoassay. Further, the number of neonatal mice, their weight and body length parameters have been assessed. Parturient mothers (n=88) and their newborn babies followed up at the Clinic of Obstetrics and Gynecology of Pavlov University took part in the clinical investigations. Concentration of BDNF, GDNF, neuron-specific enolase (NSE), and hypoxia-inducible factor (HIF-1β) in the fetal cord blood has been determined by ELISA. The obtained data were retrospectively compared with cardiotocography, dopplerometry, presence of meconium-stained amniotic fluid and the neonate state at birth, assessment according to the Apgar score, and the course of adaptation period. Results Chronic hypobaric hypoxia in pregnant mice in trimester I and II resulted in the significant decrease of BDNF and GDNF level, decrease in the number of embryos, and in significant changes in weight/height characteristics of the newborn pups. According to the clinical observations, an increased expression of the neurotrophic factors BDNF, GDNF provides protection to a neonate even if hypoxia factors are present and realized. A low content of BDNF and GDNF was observed in the group of infants with a high risk of developing unfavorable hypoxic damaging effects. Conclusion The protective role of BDNF and GDNF in the regulation of fetal homeostasis in chronic hypoxia has been established experimentally and clinically.