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.

Effect of Extracellular Vesicles Formed by Multipotent Mesenchymal Stromal Cells on Irradiated Animals.

C57Bl/6J mice were exposed to γ-radiation in a dose of 7.5 Gy. A week later, the experimental group received intravenous injection of extracellular vesicles isolated from the culture medium of human bone marrow multipotent mesenchymal stromal cells. Changes in the physiological parameters of animals were assessed by laser correlation spectroscopy, histological examination, cytometry, and by differential leukocyte count. In 3 and 6 weeks, the parameters of the experimental group occupied an intermediate position between the intact and irradiated groups or did not differ significantly from the parameters of the intact group. The rate and efficiency of recovery varied at different levels of organization. Reduction of damage caused by irradiation in a sublethal dose at different levels of organization of experimental animals was shown.

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.

[Dynamics of osteogenesis associated with inoculation of autologous stromal cells from rat adipose tissue (experimental-morphological study)].

Experiment was evaluated on 40 male Wistar rats. On the experimental model of mandible injury, bone autologous graft from tibia was placed on the surface of mandible (host bone). In the main experimental group, consisting of 20 animals, autologous rat adipose-derived stromal cells (ADSCs) were inoculated in space between autograph and host bones. ADSCs were not inoculated in the group of comparison. In experimental group with inoculated cells, the formation of a new fibroreticular bone structures in space between autograph and host bone was observed. These structures further underwent secondary reorganization and differentiation during the process of remodeling. As a result of the conducted study it was shown that in the experimental group by the day 180, statistically significant reduction of the area occupied by an immature fibroreticular bone took place. The reported phenomenon could be explained as a result of decline of the number of active cells in the population of inoculated ADSC, which is in consent with theory of limited cell division number due to telomeres shortening, described by Hayflick L. and Moorhead P.S. (1961).

[Dynamics of osteogenesis after inoculation of autogenic mesenchymal stem cells of adipose tissue].

Experiment was conducted on 40 rats of Wister line. On the artificially reproduced experimental model autogenic mesenchimal stem cells (MSC) of adipose tissue were inoculated in space between bone autograph of tibia and mandible. MSC wasn't inoculated in the comparison group. Formation of a new bone substance in space between an autograph and mandible bone was observed. It was clear that after 120 days (180 days), there was a statistically significant decline of the area occupied by an immature fibroreticular bone. Described phenomenon, presumably, could be explained as a result of decline of the number of active cells in the population of inoculated MSC according to phenomenon of limited number divisions of cells on telomeres, described by Hayflick L. and Moorhead P.S.

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.

[Influence of transplantation of autogenic mezenhymal stem cells from the fatty tissues on osseogenic process (experimental research)].

In experiments on 48 rats of line Wistar, divided at 2 groups (first--basic group and 2-d group--group of comparison) through the use of morphological method was researched influence of mesenchymal stem cells (MSC) inoculated in the area of autogenic bone graft from tibia fixed at the surface of the jaw. The animals of group of comparison didn't get MSC. Time frames of watching: 21, 60, 120 and 180 days, 6 rats on the point of experiment in either of the two of the group. Data of this research had shown the intensification of bone formation process in the basic group of experiment under the action of the inoculation of autogenic MSC from adipose tissue, and as consequence of it, in eventual, merge of bone formations in a single entity.

[Experience of culturing anterior epithelial corneal cells from human eye ball].

Adult corneal epithelium is often exposed to environmental stress, injured and repaired by limbal stem cells. Injury of corneal epithelial layer leads to reduction of visual clarity and loss of vision. Recently it was shown that epithelial layer also contains stem cells. Obtaining cell culture of corneal epithelium will allow understanding mechanisms of cell behavior and differentiation, their metabolism and reaction on environmental stress in health and disease. Moreover, cultured corneal epithelial cells can be considered as a promising material for constructing bioartificial cornea. The aim of this study was to isolate cells of anterior corneal epithelium from human donor cornea and to study their morphological and functional characteristics in vitro. The results of our study showed the possibility of culturing epithelial cells in vitro. The observed changes in cell morphology, their flow growth character as well as active proliferation and up-regulation of mesenchymal markers expression, indicate, in our opinion, epithelial-mesenchymal transition taking place in long-lasting culture of human anterior corneal epithelial cells. The obtained cultures can be used for further studies of pathological processes taking place in cells during drugs testing or controlling the phototoxic effect of different types of emission.

[The stimulation of reparative regeneration in the treatment of fractures of the extremities with the use of new biotechnologies].

The paper presents the results of a study of Russian and foreign authors on modern methods of stimulation of reparative regeneration of bone tissue. Highlight general principles for the construction and use of bioplastic materials. An attempt to classify the most famous of biomaterials based on their physico-chemical and biological properties, as well as identified trends in the further development of tissue engineering and its importance in traumatology and orthopedics.

[Dynamics of formation of bone tissue in rats under the influence of new xenotransplantat "Norian"].

In experiments on rats CD comparative studies have been carried out which is the effect of osteoplastic material NORIAN CRS, the rate of bone regeneration after mandibular dental implants (titanium screw) and the combined effect of these processes NORIAN CRS protein and protein (Emdogein). Functionality and maturity of the newly formed bone tissue was evaluated by histological and biochemical methods. The results suggest that the use of osteoplastic material NORIAN CRS is the 120 days leading to almost complete regeneration of bone tissue. It was also found that the combined use of NORIAN CRS and protein Emdogein impractical because adding Emdogein not accelerate bone regenerative process, but in the early postoperative period may cause an inflammatory reaction.

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.