[Closing of nasal septum perforation using adipose stromal vascular fraction: an experimental study]. / Zakrytie perforatsii peregorodki nosa s primeneniem stromal'no-vaskulyarnoi zhirovoi fraktsii: eksperimental'noe issledovanie.

Nasal septal perforation (NSP) is a complex problem in otorhinolaryngology, which leads to impaired nasal breathing and dryness in the nose. This reduces the patient's quality of life and leads to psychological discomfort. The treatment of nasal septum perforation is selected taking into account the clinical manifestations, perforation parameters and general condition of the patient. Currently, a large number of different surgical methods have been described in order to closing the defect of nasal septum. To date, there is no universally accepted method for closing NSP, which stimulates the search and development of new treatment options. OBJECTIVE: Under experimental conditions, to study a new method for closing nasal septum perforation using a collagen scaffold together with adipose stromal vascular fraction containing multipotent mesenchymal stromal cells. MATERIAL AND METHODS: The experiment was carried out on a model of nasal septum perforation in 24 male rabbits divided into four groups, depending on the construct, implanted into the defect zone: the 1st group was the control group - without the introduction of implantation material; the 2nd group - collagen scaffold without adipose stromal vascular fraction; the 3rd group - collagen scaffold with xenogenic adipose stromal vascular fraction; the 4th group - collagen scaffold with allogeneic adipose stromal vascular fraction with further dynamic evaluation of endoscopic control on day 14, after 1 month, 3 months, and 6 months. At month 6, the animals were removed from the experiment, followed by morphological examination in color with hematoxylin and eosin, as well as safranin and methyl green. RESULTS: As a result of the experiment using adipose stromal vascular fraction of allogeneic and xenogenic origin, closing of perforation of the nasal septum of a rabbit for 3 months of dynamic endoscopic control, as well as according to morphological research, was demonstrated. CONCLUSION: Our study showed that the use of adipose stromal vascular fraction containing not only endothelial cells and pericytes, but also multipotent mesenchymal stromal cells in combination with a collagen scaffold closes the perforation of the nasal septum in a rabbit, without increasing the risk of violations of habitual vital activity.

Regulatory network of competitively interacting RNAs and effectiveness of rectal tumors radiotherapy.

BACKGROUND: Currently, rectal tumors radiotherapy effectiveness reaches an acceptable level only in a small number of patients (they have a complete clinical response), which is associated with the formation of malignant cells radioresistance. A comprehensive study that integrates various epigenetic parameters would explain a number of molecular mechanisms of rectal tumor cells radioresistance and identify new bio-markers. In the last decade, using high-through-put sequencing, the competitively interacting RNAs regulatory network (long non-coding RNAs, miRNAs and mRNAs) has been shown. PURPOSE: The aim of the study was to analyze the features of competitively interacting RNAs regulatory network functioning in patients with rectal cancer who are radioresistant and sensitive to radiotherapy. The study was performed on 500 patients with dia-gnosed rectal cancer. Radiotherapy was performed on a Novalis TX linear particle accelerator according to the standard protocol (single focal dose 2.4 Gy, total focal dose 54.0 Gy). Total RNA preparations were isolated from paired bio-psy fragments of tumor and non-tumor tissues of the rectum (obtained by video-colonoscopy). The relative abundance of mRNA, miRNA and lncRNA transcripts was assessed by the RT-qPCR method. Using bio-informatic analysis, the probability of potential interactions between the investigated mRNA, miRNA and lncRNA was determined. It has been shown that the effectiveness of radiotherapy depends on the level of miRNA (miRNA-195-5p; miRNA-4257; miRNA-5187-5p; miRNA-149-5p; miRNA-138 -1-3p; miRNA-6798-5p; miRNA-6819-5p; miRNA-4728-5p; miRNA-1249-5p; miRNA-557; miRNA-1273h-5p; miRNA-6737-5p; miRNA-6808-5p; miRNA-3202; miRNA-5195-3p; miRNA-130b-3p) and lncRNA (XIST, HELLPAR, NEAT1, AC008124. 1, LINC01089, LINC01547, and VASH1-AS1) expression, which regulate the DNA repair system (H2AX, RBBP8) and apoptosis (BCL2). CONCLUSION: A comprehensive study of competitively interacting RNAs regulatory network and radiotherapy effectiveness of rectal tumors made it possible to establish the mechanisms of radioresistance formation and its bio-markers.

Biodegradable iron-silicon implants produced by additive manufacturing.

Due to many negative and undesirable side effects from the use of permanent implants, the development of temporary implants based on biocompatible and biodegradable materials is a promising area of modern medicine. In the presented study, we have investigated complex-shaped iron-silicon (Fe-Si) scaffolds that can be used as potential biodegradable framework structures for solid implants for bone grafting. Since iron and silicon are biocompatible materials, and their alloy should also have biocompatibility. It has been demonstrated that cells, mesenchymal stromal cells derived from the human umbilical cord (UC-MSC) and 3T3, were attached to, spread, and proliferated on the Fe-Si scaffolds' surface. Most of UC-MSC and 3T3 remained viable, only single dead cells were observed. According to the results of biological testing, the scaffolds have shown that deposition of calcium phosphate particles occurs on day one in the scaffold at the defect site that can be used as a primary marker of osteodifferentiation. These results demonstrate that the 3D-printed porous iron-silicon (Fe-Si) alloy scaffolds are promising structures for bone grafting and regeneration.

Regenerative Potential of a Suspension and Spheroids of Multipotent Mesenchymal Stromal Cells from Human Umbilical Cord on the Model of Myocardial Infarction in Rats.

Regenerative potential of multipotent mesenchymal stromal cells from the human umbilical cord (MMSC-UC) in the suspension and spheroid form was revealed during the progression of experimental small focal myocardial infarction in rats. In isoproterenol-induced myocardial infarction, foci of necrosis and inflammatory infiltrate and at later terms fibrosis foci were found mainly in the left ventricle of rat heart. In rats receiving MMSC-UC, destructive changes in the myocardium, fibrous scars, and inflammatory process were less pronounced. MMSC-UC also contributed to normalization of the morphofunctional parameters of the heart. Spheroids exhibited higher efficiency in comparison with cell suspension.

Influence of Fucoxanthin on Proliferative Activity of Human Melanocyte Culture.

We studied the effect of algae pigment fucoxanthin on proliferative activity of melanocyte culture from human skin. Fucoxanthin in high concentrations can be cytotoxic, which was confirmed by changes in melanocyte morphology and a decrease in their proliferative activity.

Digging deeper: structural background of PEGylated fibrin gels in cell migration and lumenogenesis.

Fibrin is a well-known tool in tissue engineering, but the structure of its modifications created to improve its properties remains undiscussed despite its importance, e.g. in designing biomaterials that ensure cell migration and lumenogenesis. We sought to uncover the structural aspects of PEGylated fibrin hydrogels shown to contribute to angiogenesis. The analysis of the small-angle X-ray scattering (SAXS) data and ab initio modeling revealed that the PEGylation of fibrinogen led to the formation of oligomeric species, which are larger at a higher PEG : fibrinogen molar ratio. The improvement of optical properties was provided by the decrease in aggregates' sizes and also by retaining the bound water. Compared to the native fibrin, the structure of the 5 : 1 PEGylated fibrin gel consisted of homogenously distributed flexible fibrils with a smaller space between them. Moreover, as arginylglycylaspartic acid (RGD) sites may be partly bound to PEG-NHS or masked because of the oligomerization, the number of adhesion sites may be slightly reduced that may provide the better cell migration and formation of continuous capillary-like structures.

[Case of a Сombination of Lupus Erythematosus, Antiphospholipid Syndrome and Myocardial Infarction].

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown etiology characterized by a wide range of clinical manifestations with damage to various organs and systems of the body. There are bad prognostic factors for SLE: damage to the heart, kidney, central nervous system, the development of hematological crises and secondary antiphospholipid syndrome. A number of authors consider systemic lupus erythematosus a "new" risk factor for atherosclerosis. The overall risk of myocardial infarction (MI) in patients with SLE is 10 times higher than in the general population. The article presents clinical case report of the development of myocardial infarction in a woman with SLE, receiving therapy for secondary antiphospholipid syndrome.

Induction of Vasculo- and Osteogenesis in Spheroids Formed by Adipose-Derived Stromal Cells.

Vascularization of bioengineered bone tissue constructs remains a challenging problem of regenerative medicine. Spheroids generated in 3D culture of adipose-derived stromal cells supplemented with inducing factors demonstrate stable characteristics and express of mesenchymal, endothelial, and osteoblasts markers, and represent a prototype of vascularized microtissue. Adipose-derived stromal cells spheroids induced to both angio- and osteogenic differentiation can be used in development of new innovative technologies for in vitro fabrication of vascularized bioequivalents for repair of large bone defects.

[Prognostic markers for the development of adverse outcomes in patients with chronic obstructive pulmonary disease and chronic heart failure].

AIM: To identify markers of adverse outcomes in patients with a combination of chronic obstructive pulmonary disease (COPD) and chronic heart failure (CHF). MATERIALS AND METHODS: 35 patients with COPD (without an anamnesis of coronary heart disease), 68 patients with COPD and CHF, 28 patients with CHF of ischemic genesis who were on treatment at the State Regional Clinical Hospital of Saratov were examined. The levels of the N-terminal fragment of the natriuretic peptide, galectin-3, the highly sensitive C-reactive protein, the proteins that bind fatty acids, the stiffness parameters of the arterial wall were determined; echocardiography was performed, calculated the index of comorbidity of Charlson. A year after entering the study, patients or their relatives were interviewed for their adverse outcomes. RESULTS: The combination of COPD and CHF is accompanied by an increase in the likelihood of the development of heart failure decompensation compared with the isolated course of COPD. The main causes of death of patients with combined pathology were respiratory failure and cardiovascular complications. Decompensation of CHF was 3,6 times more likely in patients with COPD and CHF of ischemic origin than in patients without previous myocardial infarction. The risk group the development of acute decompensation of heart failure within the next year is composed of patients with COPD and CHF having 3-4 functional classes of CHF, signs of decompensation in the small circulation, angina pectoris, past myocardial infarction. The most significant prognostic echocardiographic parameters were marked dilatation of the left auricles, reduction of the left ventricular ejection fraction less than 45%. The development of cardiovascular complications in patients with COPD and CHF is interrelated with an increase in arterial rigidity. The increase in total mortality is associated with the severity of heart failure and increased 24-hour arterial rigidity. CONCLUSION: The obtained results will allow to form high-risk groups and optimize the treatment-diagnostic process.

2D/3D buccal epithelial cell self-assembling as a tool for cell phenotype maintenance and fabrication of multilayered epithelial linings in vitro.

Maintaining the epithelial status of cells in vitro and fabrication of a multilayered epithelial lining is one of the key problems in the therapy using cell technologies. When cultured in a monolayer, epithelial cells change their phenotype from epithelial to epithelial-mesenchymal or mesenchymal that makes it difficult to obtain a sufficient number of cells in a 2D culture and to use them in tissue engineering. Here, using buccal epithelial cells from the oral mucosa, we developed a novel approach to recover and maintain the stable cell phenotype and form a multilayered epithelial lining in vitro via the 2D/3D cell self-assembling. Transitioning the cells from the monolayer to non-adhesive 3D culture conditions led to formation of self-assembling spheroids, with restoration of their epithelial characteristics after epithelial-mesenchymal transition. In 7 days, the cells within spheroids restored the apical-basal polarity, and the formation of both tight (ZO1) and adherent (E-cadherin) intercellular junctions was shown. Thus, culturing buccal epithelial cells in a 3D system allowed us to recover and durably maintain the morphological and functional characteristics of epithelial cells. The multilayered epithelial lining formation was achieved after placing spheroids for 7 days onto a hybrid matrix, which consisted of collagen layers and reinforcing poly (lactide-co-glycolide) fibers and was proven promising for replacement of the urothelium. Thus, we offer an effective technique of forming multilayered epithelial linings on carrier-matrices using cell spheroids that was not previously described elsewhere and can find a wide range of applications in tissue engineering, replacement surgery, and regenerative medicine.

Angiogenic potential of spheroids from umbilical cord and adipose-derived multipotent mesenchymal stromal cells within fibrin gel.

One of the essential goals in regenerative medicine is microvascularization which enables an effective blood supply within de novo constructed tissues and organs. In our study, we used two common multipotent mesenchymal stromal cell (MMSC) sources (subcutaneous adipose tissue and Wharton's jelly of the umbilical cord) where is a subpopulation of endothelial precursors. In the medium supplemented with VEGF, the 3D cultures of UC MMSCs and ADSCs promoted the endothelial cell differentiation. To evaluate their ability to form a capillary-like network, we encapsulated spheroids within non-modified and PEGylated fibrin hydrogels. The PEGylated hydrogel supported better the formation of multibranched cords than the pure fibrin gel. Analysis of tubule growth rate, length, and branching showed that the differentiated ADSCs had higher angiogenic potential than the differentiated hUC MMSCs. Our study can be a basis for the development of new strategies in tissue engineering and treatment of vascular diseases.

Characteristics of Trophoblasts in Long-Term Culture.

We analyzed more than 40 cytotrophoblast cultures derived from cell islets that grew from trypsinized tissue fragments of placental microvilli. Phenotypic variability of trophoblasts was demonstrated. Changes in trophoblast morphology from epithelium-like or oval cells to bipolar and spindle-shaped or twisted and then to mesenchymal-like cells as well as intensive expression of cytokeratin-7 and vimentin attested to epithelial-mesenchymal transition of trophoblasts during in vitro culturing. Analysis of the expression of specific markers in long-term trophoblast culture (≥7 passages) revealed the possibility of culture contamination with other non-trophoblast cells including fibroblasts. High risk of trophoblast culture contamination with rapidly growing cells necessitates regular control of the cultures used in fundamental studies. Our experiments confirmed the possibility of long-term culturing of cells maintaining trophoblast properties. The identity and purity of 4 trophoblast cultures free from contamination and retaining the properties of pure culture during long-term (>10 passages) culturing in vitro were confirmed.

[Cellular Model Based on Laser Microsurgery of Cell Spheroids to Study the Repair Process].

In this study, modern techniques of laser microsurgery of cell spheroids have been used to develop a new simple, reproducible model for studying the mechanisms of repair and regeneration in vitro. Nanosecond laser pulses were applied to perform a microdissection of the outer and the inner zones of the spheroids from dermal fibroblasts. To achieve effective dissection and preservation of spheroid viability, the optimal parameters were chosen: 355 nm wavelength, 100 Hz frequency, 2 ns pulse duration, laser pulses in the range of 7­9 µ J. After microdissection, we observed injury of the spheroids : the edges of the wound surface opened and the angular opening reached a value of more than 180°. As early as during the first hour after spheroid microdissection with laser radiation, the surviving cells changed their shape: cells on the spheroid surface and directly in the damaged area became rounded. One day after microdissection, the structure of the spheroids began to partially recover, the cells in the surface layers began to take the original flattened shape; debris of dead damaged cells and their fragments was gradually cleared from the spheroid composition. In the proposed model, the first data on stimulation of structure recovery of injured spheroids from dermal fibroblasts with a P199 synthetic polypeptide, which is used in cosmetology for the initiation of antiaging and regenerative effects in the skin, were received. After microdissection, recovery of the spheroids structure with a few surface layers of flattened imbricated arranged cells and polygonal cells of the inner zone in the presence of P199 peptide was faster than in the control group, and was completed within 7 days, presumably due to the remodeling of the survived cells.

[The cross-plastic correction of the soft tissue defect on the neck with the use of a pectoral flap harvested from the opposite side]. / Perekrestnaya plastika defekta myagkikh tkanei shei pektoral'nym loskutom s protivopolozhnoi storony.

The authors describe a patient presenting with initially multiple metachronous cancer spreading over the right-handed side of the mouth floor and the right-handed part of the lower jaw. The patient who underwent cross-plastic surgery for the correction of the extensive soft tissue defect on the neck with the use of the musculocutaneous pectoral flap with the axial blood supply is described. The observation during the 14 year follow-up period confirmed the good functional outcome of the treatment.

Laser-based technique for controlled damage of mesenchymal cell spheroids: a first step in studying reparation in vitro.

Modern techniques of laser microsurgery of cell spheroids were used to develop a new simple reproducible model for studying repair and regeneration in vitro Nanosecond laser pulses (wavelength 355 nm, frequency 100 Hz, pulse duration 2 ns) were applied to perform a microdissection of the outer and the inner zones of human bone marrow multipotent mesenchymal stromal cells (BM MMSC) spheroids. To achieve effective dissection and preservation of spheroid viability, the energy of laser pulses was optimized and adjusted in the range 7-9 µJ. After microdissection, the edges of the wound surface opened and the angular opening reached a value of more than 180°. The destruction of the initial spheroid structure was observed in the wound area, with surviving cells changing their shape into a round one. Partial restoration of a spheroid form took place in the first six hours. The complete structure restoration accompanying the reparative processes occurred gradually over seven days due to remodelling of surviving cells.

Pathophysiological and molecular mechanisms of extracellular matrix protein resorption during skin aging, and the ways to their restoration.

The article is a short review of the most studied molecular mechanisms leading to skin aging. It considers mechanisms of cellular aging, oxidative stress, development of chronic inflammation, as well as synthesis and degradation of extracellular matrix proteins. The review also contains examples of extracellular matrix restoration using cell and pharmacological technologies.

The technology of obtaining multipotent spheroids from limbal mesenchymal stromal cells for reparation of injured eye tissues.

It is known that stem and progenitor cells open new possibilities for restoring injured eye tissues. Limbal eye zone, formed mainly by derivatives of neural crest, is the main source of stem cells for regeneration. The current study considers development of innovative technology for obtaining 3D spheroids from L-MMSC. It was shown that under 3D conditions L-MMSC due to compactization and mesenchymal-epithelial transition self-organize into cellular reparative modules. Formed L-MMSC spheroids retain and promote undifferentiated population of stem and progenitor limbal cells, as supported by expression of pluripotency markers - Oct4, Sox2, Nanog. Extracellular matrix synthetized by cells in spheroids allows retaining the functional potential of L-MMSC that are involved in regeneration of both anterior and, probably, posterior eye segment.

3D-technology of the formation and maintenance of single dormant microspheres from 2000 human somatic cells and their reactivation in vitro.

We developed an original reproducible 3D-technology for preparation of single dormant microspheres consisting of 2000 somatic cells. The dynamics of microsphere assembly from mesenchymal and epithelial cells of retinal pigment epithelium was traced using time-lapse microscopy: formation of a loose aggregate over 24 h followed by its gradual consolidation and formation of a compact viable microsphere with a diameter of 100-150 µ by day 7. The cell number in the formed microspheres remains unchanged. Reactivation observed upon fusion of epithelial and/or mesenchymal microspheres results in the formation of a united compact microtissue. The fusion dynamics reproduces spherogenesis irrespective of the initial amount of co-cultured microspheres. Reactivation via two-step induced angiogenesis opens new prospects for production of vascularized microspheres and microtissues.

[Vascular remodeling indicators and their impact on prognosis in patients with chronic heart failure].

AIM: To estimate vascular remodeling indicators and their impact on prognosis in patients with chronic heart failure (CHF). SUBJECTS AND METHODS: The study enrolled 128 patients with prior myocardial infarction and CHF and 40 individuals as a comparison group. Endothelium-dependent and -independent vasodilation (EDV and EIDV) was studied in the reactive hyperemia (RH) test and nitroglycerin (NTG) test. Carotid artery (CA) intima-media thickness (IMT) and great arterial rigidity were studied. RESULTS: As compared to the control subjects, the patients with CHF were found to have EDV and EIVD reductions that were more pronounced in those with Functional Class IV CHF, more CA IMT, and abnormal arterial rigidity. There were relationships of EDV and EIDV to CA IMT (r = -0.30; p = 0.02) and aortic pulse wave velocity (APWV) (r = -0.30; p = 0.05). Multivariate analysis showed that the risk for cardiovascular events (CVE) in the CHF patients with an APWV of > 12 m/sec during a year was 2.02 times higher than in those with an APWV of < or = 12 m/sec (relative risk 2.02, 95 confidence interval, 1.01-4.03). CONCLUSION: APWV is an independent risk factor of CVE in patients with CHF.

[Prognostic significance of cardiac rhythm variability in patients with chronic heart failure].

Prognosis of chronic heart failure (CCF) is a challenging clinical problem. The role of heart rhythm variability (HRV) in CCF pathogensis is poorly known. This study was aimed at estimating the possibility of using HRV for prognosis of CCF. It included 110 patients (mean age 55.1 +/- 6.4 yr) with CCF symptoms after myocardial infarction. HRV analysis was based on 5 minute records. During the first year; 44 patients developed cardiovascular complications, 20 aggravation of CCF 5 unstable angina, 7 repeated infarction, 9 died. The low-frequency component (LF) of the spectrum was inversely related to age, HR, NT-proBNP, CRP and left ventricular election fraction. Based on the low-frequency component median, the patients were allocated to 2 groups (less than 5.2 ln ms2 and 5.2 ln ms2 or more). Comparison of survival curves by the Kaplan-Meier method showed that at LF < 5.2 ln ms2 the probability of CCF decompensation (p = 0.03), secondary infarction (p = 0.007), lethal outcome (p = 0.03) is significantly higher than at LF of 5.2 ln ms2 or more. It means that LF < 5.2 ln ms is an independent risk factor of CCF decompensation, repeated myocardial infarction, amd lethal outcome.