[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.

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.

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.

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.

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.

Behavior of Transplanted Multipotent Cells after in Vitro Transplantation into the Damaged Retina.

The use of stem cell technologies in retinal defect reparation therapy has produced beneficial results. Nowadays, numerous protocols exist which provide a neural differentiation of the stem cells transplanted into the retina. However, questions concerning the functional replacement of the missing retinal neurons by transplanted cells thus far remain unanswered. The organotypic culture protocol was used in this study in order to prove the possibility of transdifferentiation of bone marrow stromal cells (MMSCs) and neural stem/progenitor cells (NSPCs) from EGFP-positive mice and the functional integration of these cells. This technique enables a detailed characterization of cell behavior post-transplantation. Using atomic force microscopy, we reliably demonstrated the difference (p < 0.01) between the thickness of the outgrowths formed by glial and endothelial retina cells and the thickness of neurites and neuro-like transplanted MMSC outgrowths. MMSCs are also shown to form synapses up to 2.5 ± 0.06 µm in diameter on day 4 after the transplantation. Following electrical stimulation (20V, 0.5Hz, 200ms), clear depolarization of retinal neurons and their outgrowths is detected. It is shown that some of these GFP+ MMSCs, which changed their morphology after the transplantation in retinal explants to neuro-like MMSCs, are capable of depolarizing after exogenous stimulation.

[Experimental study of safety in application of the Iris Medical IQ 810 diode laser in clinical treatment of age-related macular degeneration].

During the study of diode laser radiation effect in micropulse mode on culture cells of human retinal pigment epithelium it was revealed that the quota of dead cells was a minimum. Besides, a certain conformity between dead cells quota and parameter characteristics of laser radiation. Based on the performed experimental study it was revealed that for a work using the Iris Medical IQ 810 diode laser in the micropulse mode following parameters: duration of pulse set--300ms, duration of function--9.1%, power--750mW are safe for retinal pigment epithelium cells. Rationales of safety in application of the infrared diode laser radiation in micropulse mode in clinic for treatment of age-related macular degeneration (AMD) exampled by cell culture of human retinal pigment epithelium.

[The effect of antisense oligonucleoties specific to the harakiri mRNA on spontaneous and induced defects of mouse preimplantation embryo development].

The effect of antisense oligonucleotides specific to mRNA of the proapoptotic gene harakiri (Hrk) on the development of mouse SAMP1 (senescence-accelerated mouse prone) and (C57BL/6J x DBA/2J)F1 preimplantation embryos cultured in vitro was investigated. The SAMP1 mice are characterized by genetically determined decrease of fertility along with the highly frequent perturbations of embryonic development. Reproduction indices of the (C57BL/6J x DBA/2J) hybrids lie within the normal range. Because of this, preimplantation abnormalities in this line were induced by the action of proapoptotic agent bleomycine. It was demonstrated that antisense inhibition of the Hrk expression had no effect on the frequency of genetically determined abnormalities of early embryonic development in SAMP1 mice. In case of induced abnormalities, addition of oligonucleotides specific to mRNA of proapoptotic Hrk gene influenced the number of abnormalities, and at the same time, improved the quality of survived embryos via increasing the blastocyst hatching.

[Ultrasonographic guidance in diagnostic and therapeutic interventions in diseases of the internal organs]. / Ul'trazvukovoi metod kontrolia za diagnosticheskimi i lechebnymi vmeshatel'stvami pri zabolevaniiakh vnutrennikh organov.

A hundred fifty four invasive diagnostic and therapeutical interventions were made in patients with diseases of the abdomen and retroperitoneal space under ultrasonographic guidance. Various biopsies were performed in 139 patients; positive results were achieved in 120 (86.3%) cases. In 15 patients, diagnostic biopsies were combined with therapeutical interventions, such as aspiration of cysts in the liver (n = 3) and kidney (n = 2); drainage of abscesses in the abdomen (n = 5) and liver (n = 5). Fourteen patients with mechanical jaundice caused by extrahepatic bile duct tumors or pancreatic head cancer underwent percutaneous transhepatic cholecystocholangiography followed by external drainage. Percutaneous transhepatic drainage of the gallbladder was made in 1 patient with acute cholecystitis.

[MRI diagnosis of soft tissue tumors]. / MR-tomograficheskaia diagnostika opukholei miagkikh tkanei.

MRI findings of 13 patients with soft tissue tumors (STT) are presented. There are were abnormalities, such as primary STTs of the hip in 5 patients, the back in 1, and the neck in 1, STT relapses of the hip in 2 and those of the back in 1. Two patients had chronic hip STT hematomas and 1 had hip STT metastatic melanoma. The diagnosis was verified in 11 cases (in 10 cases at surgery and 1 at needle biopsy). MRI makes it possible to define the accurate sizes of the tumor, its structure and relationship to its adjacent tissues, which is important in choosing at treatment policy, the type and scope of a surgical interventions.

[Puncture methods of the diagnosis and treatment under the control of US, CT and X-ray video image]. / Punktsionnye metody diagnostiki i lecheniia pod kontrolem UZ, KT i rentgenotelevizionnogo izobrazheniia.

Exploratory and therapeutical punctures were made in 454 patients under the control of ultrasound, CT, and X-ray teleimage. Diagnostic biopsies established the cytological and histological nature of diseases in 67.6% of cases, positive ultrasound-, CT-, and X-ray teleimage-controlled biopsies being 76.3, 64.9, and 71.4%, respectively. Beneficial effects of therapeutical interventions were obtained in 89.5% of patients.