We studied the effect of conditioned media (CM) from cultivated bone marrow stromal cells grown at 10% O2 on extracellular matrix and vascular component in the healing zone after surgical incision of the uterine wall in Sprague-Dawley rats (n=17). Control group (n=10) received no treatment. On days 5 and 15 after the surgery, the expression of CoL1a1, CoL4a, MMP9, TIMP1, and FGF2 genes was evaluated and a morphological study was carried out. On day 5, CoL1a1 expression, CD34+ cell content, and the area of newly formed tissue were lower in the experimental group. On day 15, the expression of MMP9, TIMP1, FGF2, and CoL1a1 genes in the control group was lower, while CD34+ cell content and area of healing zone were higher. Thus, the application of CM reduced the damage area and accelerated the recovery process after surgical full-thickness incision of the uterine wall.

Purpose: to evaluate in experiment the dynamics and intensity of vascular sprouting in the donor cornea during keratoplasty against the background of pre-existing neoangiogenesis when using a conditioned medium of mesenchymal stem cells.Materials and methods. The object of the study was 15 rabbits (10 recipient rabbits, 5 donor rabbits) of the gray Chinchilla breed weighing 2.5–3.2 kg. The experiments were carried out in two stages. At the first stage, 10 animals were simulated with a unilateral thermal burn of the peripheral zone of the cornea with limbal involvement. Penetrating keratoplasty was performed using standard technology on the 15th day of the experiment. At the second stage, the recipient rabbits were divided into experimental and control groups. 5 rabbits, 5 eyes, in the control group and 5 rabbits, 5 eyes, in the main group. In the main group, instillations of paracrine factors of mesenchymal stem cells were used as postoperative treatment from the 1st to the 30th knocks four times. In the control group, treatment was carried out by instillation of antibiotics (Oftaquix) and keratoprotectors (Korneregel) three times for 30 days. Research methods included biomicroscopy, photographic recording of the anterior segment of the eye, examination using a Pentacam AXL device, and histological examination. The observation periods were 1st, 3rd, 7th, 14th, 30th day.Results. The results of postoperative treatment at the second stage of the experiment, as well as the results of histology, showed a pronounced antiangiogenic effect of the conditioned medium of mesenchymal stem cells. This was confirmed by the absence of pronounced vascularization of the donor cornea, the attenuation of the inflammatory reaction and the formation of a scar on the 30th day of drug instillation. In the control group, graft disease developed from the seventh day.Conclusion. The results obtained during the work seem promising. The use of the conditioned medium of mesenchymal stem cells demonstrated a sufficiently pronounced effectiveness compared with standard drug therapy in the postoperative period after high-risk end-to-end keratoplasty in experimental animals performed on a model of post-burn corneal neovascularization.

A mesenchymal stem cell (MSC)-derived conditioned medium (CM) was tested for effects on the severity of the systemic inflammatory response induced by acetaminophen (APAP), including its long-term influence on hepatic tissue. A CM fraction of <30 kDa showed a maximum protective effect. Proteins of the fraction reduced the systemic inflammatory response and liver tissue fibrosis long after the toxin administration. Liver tissues from experimental animals were examined, and reduction of granulocytic infiltration was found to correlate with the degree of hepatic parenchymal necrosis, which was confirmed by the cytolysis enzyme level. The <30-kDa fraction increased the spontaneous and decreased the phorbol 12-myristate 13-acetate (PMA)-induced intracellular production of reactive oxygen species (ROS). Expression of the macrophage migration inhibitory factor (MIF) and thioredoxin significantly increased as compared to controls during increased spontaneous ROS production in neutrophils.

В связи с увеличением числа заболеваний, в основе которых лежит развитие новообразованных сосудов, в последние десятилетия возрос интерес к проблеме патологического ангиогенеза в различных тканях глаза. Традиционные консервативные методы лечения неоваскуляризации роговицы на данный момент недостаточно эффективны. Возможным путем решения проблемы неоваскуляризации роговицы может быть использование паракринных факторов, выделяемых мезенхимальными стволовыми клетками в процессе культивирования в кондиционированную среду (КС). Целью исследования является изучение влияния пептидов КС на сосудистое бельмо, которое сформировалось после химического ожога глаз крыс в эксперименте, и оценка влияния КС на функциональную активность нейтрофилов. Методы исследования. Исследование было выполнено на 48 крысах (48 глаз) в несколько этапов. На первом этапе всем животным был смоделирован химический ожог роговицы. На втором этапе проводилось лечение ожогов роговицы. В зависимости от способа лечения на втором этапе была сформирована контрольная группа 22 крысы (22 глаза) и опытная группа 22 крыс (22 глаза). Опытная группа была разделена на 2 подгруппы. В первой подгруппе было 11 крыс (11 глаз), которым в качестве лечения применялись пептиды КС общей фракции. Во второй подгруппе также было 11 крыс (11 глаз), в качестве лечения применялся пептиды КС фракцией до 30 кДа. В контрольной группе для лечения ожогов мы применяли культуральную среду. Результаты. По результатам офтальмоскопии и гистологических исследований на 14-е и 30-е сутки в контрольной группе животных динамики не наблюдалось. Выявлялись множественные новообразованные сосуды роговицы (преимущественно крупные сосудистые стволы) со стромальными помутнениями в их проекции. Эпителий роговицы был не изменен, однако, сама роговица была истончена. Наблюдалось сформированное васкуляризированное бельмо роговицы: субэпителиальная фиброплазия и неоваскуляризация роговицы. В опытной группе животных к 14-м суткам инстилляций в конъюнктивальную полость пептидов КС офтальмоскопически мы наблюдали уменьшение количества новообразованных сосудов, помутнения роговицы исчезли в проекции запустевших сосудов. На 30-е сутки лечения в опытных группах животных у большинства крыс новообразованных сосудов обнаружено не было, сохранялись лишь отдельные сосудистые стволы, которые ранее образовывали мощные анастамозы с другими новообразованными сосудами. По результатам хемолюминесцентного анализа показано, что фракция до 30 кДа угнетает функциональную активность нейтрофилов, в том числе за счет изменения активности внутриклеточных мембранных ферментов, отвечающих за передачу рецепторных сигналов внутрь клетки. Заключение. Полученные результаты свидетельствуют о перспективности применения препарата раствора пептидов для лечения такой сложной патологии, как неоваскуляризация роговицы. In recent decades, due to increasing number of diseases associated with the development of newly formed vessels, the interest to pathological angiogenesis in eye tissues has been growing. Traditional, conservative methods for treatment of corneal neovascularization are not sufficiently effective. A possible solution may be the use of paracrine factors secreted by mesenchymal stem cells during cultivation in a conditioned medium (CW). The aim of this study was to assess the effect of CW peptides on vascular leukoma induced by chemical burn of rat eyes and to evaluate the effect of CW on the functional activity of neutrophils. Methods. The study was performed on 48 rats (48 eyes) and included several stages. At the first stage, chemical corneal burn was modeled in all animals. At the second stage, corneal burns were treated. Based on the method of treatment at the second stage, a control group (22 rats, 22 eyes) and an experimental group (22 rats, 22 eyes) were formed. The experimental group was divided into 2 subgroups. The first subgroup rats (n = 11, 11 eyes) were treated with CW peptides of the entire fraction. The second subgroup rats (n = 11, 11 eyes) received a CW peptide fraction of up to 30 kDa. In the control group, the culture medium was used as a treatment. Results. According to the results of ophthalmoscopy and histological studies on days 14 and 30, no changes were observed in the control group. Multiple newly formed corneal vessels (mainly large vascular trunks) with stromal opacities in their projection were detected. The corneal epithelium was unchanged; however, the cornea itself was thinned. A formed vascularized corneal leukoma was observed that was evident as subepithelial fibroplasia and corneal neovascularization. In the experimental group by the 14th day of CW peptide instillations into the conjunctival cavity, ophthalmoscopy showed a decrease in the number of newly formed vessels, and corneal opacities disappeared in the projection of the deserted vessels. In most of rats in the experimental groups on the 30th day of the treatment, newly formed vessels were not found; only individual vascular trunks remained, which had previously formed powerful anastomoses with other newly formed vessels. According to the results of chemiluminescence analysis, a fraction of up to 30 kDa inhibited the functional activity of neutrophils, including by changing the activity of intracellular membrane enzymes responsible for the transmission of receptor signals into the cell. Conclusion. The results showed that the peptide solution is promising for the treatment of such a complex pathology as corneal neovascularization.

Цель - изучение влияния трансплантации стромально-васкулярной фракции (СВФ), культивированных мультипотентных мезенхимальных стволовых клеток (ММСК) жировой ткани и введения паракринных факторов кондиционной среды, полученных при культивировании ММСК, на течение тяжелых местных радиационных поражений кожи. Методика. Крыс инбредной линии Wistar-Kyoto облучали на рентгеновской установке в дозе 110 Гр, при мощности дозы 20,0 Гр/мин. Напряжение на трубке 30 кВ, ток 6,1 мА, фильтр Al толщиной 0,1 мм, площадь поля облучения составляла 8,5 см2. Трансплантацию клеток СВФ жировой ткани проводили двукратно в дозах 2,2 х 106 и 3,0 х 106 на 28-е и 35-е сут после облучения, соответственно. Культивированные ММСК вводили в дозах 2,8 × 106 и 3,4 × 106 на 28-е и 35-е сут после действия радиации. Паракринные факторы кондиционной среды вводили пятикратно с 1-х по 10-е сут после облучения, подкожно по 1,0 мл (общий белок 8 мг/мл). Тяжесть лучевого поражения кожи и эффекты терапии оценивали в динамике по клиническим проявлениям, с помощью планиметрии и патоморфологических методов. Результаты. Выбранный режим радиационного воздействия вызывал тяжелые лучевые поражения кожи с длительно (до 5 мес) незаживающими язвами. Площадь лучевых язв у крыс контрольной группы в период с 28-х до 84-х сут медленно уменьшалась от 2,73±0,17 см2 до 1,52±0,17 см2, а через 4 и 5 мес после облучения составляла 0,52±0,15 см2 и 0,05±0,04 см2, соответственно. Во всех опытных группах после 84-х сут отмечалась тенденция к снижению площади лучевых язв, а через 4 мес после облучения уменьшение площади язв и снижение тяжести поражения в опытных группах по сравнению с контролем были статистически значимыми (р < 0,05). У 70-80 % крыс в опытных группах через 4 мес после облучения наблюдалась полная эпителизация радиационных ран с образованием атрофического рубца, а в контрольной группе в этот период атрофические рубцы наблюдались лишь у 40% крыс. Заключение. Трансплантация СВФ жировой ткани или культивированных ММСК, а также введение паракринных факторов кондиционной среды способствуют усилению регенераторных процессов в пораженной ткани, и могут быть в равной степени эффективны при терапии тяжелых лучевых поражений кожи в эксперименте. Aim. To study effects of stromal-vascular fraction (SVF) transplantation, cultured multipotent mesenchymal stem cells (MMSC) of adipose tissue, and administration of paracrine factors of the conditioned MMSC culture medium on the course of severe, local radiation lesions of the skin. Methods. Wistar-Kyoto rats were irradiated with X-rays at a dose of 110 Gy and a dose rate of 20.0 Gy/min. The tube voltage was 30 kV, current - 6.1 mA, filter -0.1 mm thick A1, and irradiation field area - 8.5 cm2. Transplantation of adipose tissue SVF cells was performed twice at doses of 2.2 x 106 and 3.0 x 106 on the 28th and 35th days after irradiation, respectively. Cultured MMSC was administered at doses of 2.8 x 106 and 3,4 x 106 on the 28th and 35th days after irradiation. Paracrine factors of the conditioned medium were administered five times from the 1st to the 10th day after irradiation at a dose of 1.0 ml, s.c. (total protein, 8 mg/ml). Severity of radiation damage of the skin and effects of the therapy were followed up by clinical manifestations using planar geometry and pathomorphological methods. Results. Radiation exposure under the specified conditions caused severe radiation injuries of the skin with nonhealing ulcers that persisted for up to 5 months after irradiation. In control rats the area of radiation ulcers slowly decreased between the 28th and 84th days from 2.73 ± 0.17 cm2 to 1.52 ± 0.17 cm2, respectively, and was 0.52 ± 0.15 cm2 and 0.05 ± 0.04 cm2 at 4 and 5 months after irradiation. In all experimental groups, the healing was more intensive after the 84th day with a tendency to decrease the area of radiation ulcers; at 4 months after irradiation, statistically significant decreases in the area of ulcers and severity of lesions were observed in the experimental groups compared to the control (p <0.05). At four months after irradiation, 70-80% of rats in the treatment groups showed complete epithelialization of radiation wounds with formation of an atrophic scar whereas in the control group during the same period, atrophic scars were observed only in 40% of rats. Conclusion. Transplantation of adipose-derived SVF or cultured adipose tissue MMSC as well as administration of paracrine factors of the conditioned medium contributed to the enhancement of regenerative processes in the affected tissue and can be equally effective in the treatment of severe experimental radiation lesions of the skin.

Background. In case of a corneal burn injury, cell transplantation into the damaged area must be performed within the first 12 hours, which makes it impossible to use autologous stem cells. One solution to this problem may be the use of peptides, derived from cultured stem cells in the treatment and prevention of complications in a burn eye disease. Aims: To study the dynamics of corneal tissue repair under the influence of a peptide solution on a corneal thermal burn model.Materials and methods. The study included 20 rabbits (20 eyes) of the gray Chinchilla breed weighing from 2.5 to 3.2 kg with a corneal thermal burn model. Depending on the method of treatment used, the animals were divided in two groups of 10 rabbits (10 eyes). In the experimental group, instillations of a peptide solution were used to treat corneal thermal burns; in the control treatment was carried out with a solution of moxifloxacin and gel “Solcoseryl”. On the 1st, 3rd, 7th, 14th, 30th days in each group, two animals were sacrificed to conduct a morphological study of the cornea. Results. In the experimental group, by the 30th day, according to a histological study, the inflammatory process was completed both on the surface and inside the cornea, with a tendency to restore its normal structure. In the control group of animals, significantly longer periods of corneal recovery and preservation of inflammation, despite the received therapy, were observed.Conclusions. The use of the peptide preparation is promising in the treatment of corneal thermal burn. Further research is needed in this area.

The effect of different polyoxyethylene–polyoxypropylene triblock copolymers, their concentration, and mode of action on the loading of poly-(lactic-co-glycolic acid) copolymer-based microparticles containing such medicinal agents as radachlorin (chlorin e 6 ) or ethidium bromide in mesenchymal stem cells was studied. It has been shown that medicinal agents encapsulated inside microparticles affect the loading of these particles in the cytoplasm of mesenchymal stem cells. The number of cells that absorbed the particles with chlorin e 6 is approximately two times lower than that in the experiments with ethidium bromide. It has been shown that pretreatment of microparticles with triblock copolymers is more efficient for loading them in cells compared with simultaneous introduction of triblock copolymers and particles into the culture medium. Treatment of ethidium bromide-containing microparticles with triblock copolymers is not efficient for their loading in mesenchymal stem cells compared to the control. The exception is Pluronic 123; when particles are treated with it at concentrations of 1 and 2%, the loading of particles in cells increases compared to the control by factors of approximately 11 and 5, respectively. For particles with chlorin e 6 , their pretreatment with triblock copolymers at a concentration of 4% is most efficient; the loading of the pretreated particles in cells is increased by factors of approximately 3 to 11.

Data on the efficiency of low-molecular peptide preparations, selected from cultivated stem cells of marrow, in the treatment of acute kidney insufficiency in experimental animals are resulted. It is demonstrated that the use of peptide preparations reliably promotes survivability of experimental animals suffering from acute kidney insufficiency.

Data on the efficiency of low-molecular peptide preparations, selected from cultivated stem cells of marrow, in the treatment of acute kidney insufficiency in experimental animals are resulted. It is demonstrated that the use of peptide preparations reliably promotes survivability of experimental animals suffering from acute kidney insufficiency.

The purpose of this review was to systematize data of many studies and observations of paracrine factors of MSC and their biological effects. Also a brief description of main groups of cytokins was revealed in the review.

Purpose: This study focuses on the development of the method of introduction of magnetic microparticles in the cytoplasm of HEK-293 cell line with their subsequent fixation under the retina of the eye.Materials and Methods. Magnetic particles (d = 2,8 mm) were treated with pluronic and injected into the cytoplasm of HEK-293 cell line, expressing GFP. The surgery was made under general anesthesia. HEK-293 containing magnetic particles were injected into the subretinal space of rabbit eyes (eyes 96, 48 rabbits) using original dosing device. In the experimental group (48 eyes, 24 rabbits) we fixed episcleral magnetic implant to hold cells in local place. In the control group (48 eyes, 24 rabbit) magnetic implant was not fixed. After the surgery all animals were examined using biomicroscopy, ophthalmoscopy with photographic recording, ultrasound, computed tomography and morphological study in certain terms (1, 3, 5, 7, 14, 21 day and 1 month).Results: The introduction of the magnetic particles into the cytoplasm of HEK 293 cell line has no effect on cell viability. HEK-293 containing magnetic particles remains in the place of injection during 21 days in rabbit eyes, where the magnetic implants were fixed (in control group during 3 days). Conclusions: Using of cells containing magnetic particles with fixation of the magnetic implant can be a promising method for cell therapy for the treatment of retinal diseases.

A composition containing culture medium conditioned by mesenchymal stem cells and mesenchymal stem cell lysate improves biochemical parameters, reduces inflammation, and stimulates regenerative processes in the liver.

Purpose: This study focuses on the development of the method of introduction of magnetic microparticles in the cytoplasm of HEK-293 cell line with their subsequent fixation under the retina of the eye. Materials and Methods. Magnetic particles (d = 2,8 mm) were treated with pluronic and injected into the cytoplasm of HEK-293 cell line, expressing GFP. The surgery was made under general anesthesia. HEK-293 containing magnetic particles were injected into the subretinal space of rabbit eyes (eyes 96, 48 rabbits) using original dosing device. In the experimental group (48 eyes, 24 rabbits) we fixed episcleral magnetic implant to hold cells in local place. In the control group (48 eyes, 24 rabbit) magnetic implant was not fixed. After the surgery all animals were examined using biomicroscopy, ophthalmoscopy with photographic recording, ultrasound, computed tomography and morphological study in certain terms (1, 3, 5, 7, 14, 21 day and 1 month). Results: The introduction of the magnetic particles into the cytoplasm of HEK 293 cell line has no effect on cell viability. HEK-293 containing magnetic particles remains in the place of injection during 21 days in rabbit eyes, where the magnetic implants were fixed (in control group during 3 days). Conclusions: Using of cells containing magnetic particles with fixation of the magnetic implant can be a promising method for cell therapy for the treatment of retinal diseases.

It has been shown that sorption of most proteins with the molecular weight lower than 200 kDa from human blood plasma on the surface of perfluorocarbon emulsion stabilized with proxanol 268 is mainly based on hydrophobic interaction, whereas sorption of immunoglobulin G is mainly the result of electrostatic interaction. The removal of lipidic components from plasma leads to an increase in the total amount of adsorbed proteins by 35%. Particularly, when lipidic components are removed, sorption of apolipoprotein AI and fibrinogen is considerably bettered as well as sorption of other proteins with the molecular weight of about 50 and 60 kDa occurs. It has been set that apolipoprotein AI in the adsorbed condition loses its capability of tryptophan fluorescence, which might be probably determined by the quenching influence of the perfluorocarbon core of nanoparticle. We think that the findings obtained also indicate considerable conformational rearrangements of this protein during adsorption. It was shown that the fluorescence of proteins with sorption on nanoparticles in emulsion based on the hydrophobic interaction is completely or partially quenched.

The adsorption abilities of the perfluorocarbon emulsion stabilized by Proxanol 268 were investigated in vitro and in vivo. In vitro, the saturation point for the blood plasma proteins was nearly reached after five minutes of incubation of the emulsion with human/rabbit blood plasma and was stable for all incubation periods studied. The decrease in volume ratio (emulsion/plasma) was accompanied by the increase in the adsorptive capacity of the emulsion with maximal values at 1/10 (3.2 and 1.5 mg of proteins per 1 mL of the emulsion, for human and rabbit blood plasma, respectively) that was unchanged at lower ratios. In vivo, in rabbits intravenously injected with the emulsion, the proteins with molecular masses of 12, 25, 32, 44, 55, 70, and 200 kDa were adsorbed by the emulsion (as in vitro) if it was used 6 h or less before testing. More delayed testing (6 h) revealed elimination of proteins with molecular masses of 25 and 44 kDa and an additional pool of adsorbed new ones of 27, 50, and 150 kDa. Specific adsorptive capacity of the emulsion enhanced gradually after emulsion injection and reached its maximum (3.5–5 mg of proteins per 1 mL of the emulsion) after 24 h.