Background: The search for an effective therapy for local radiation injuries (LRI) is urgent; one option is mesenchymal stem cells (MSC) derived from the placenta and their conditioned medium for the regenerative processes of the skin. Methods: We used 80 animals, randomly assigned to four groups: control (C) animals that did not receive therapy; control with the introduction of culture medium concentrate (CM); introduction of MSCs (PL); introduction of CMPL. LRI modeling was performed on an X-ray machine at a dose of 110 Gy. Histological and immunohistochemical tests were performed. Results: On the 112th day, the area of the open wound surface in the CMPL group was 6.7 times less than in the control group. Complete healing of the open wound surface of the skin in the CM group was observed in 40%, in CMPL 60%, in the PL group 20%, and in the C group there were no animals with a prolonged wound defect. A decrease in inflammatory processes was observed in the CMPL group. Conclusions: the use of a concentrate of conditioned MSCs (CMPL group) in severe LRI in laboratory animals accelerates the transition of the wound process to the stage of regeneration and epithelization.
Purpose: To study the regeneration processes in the treatment of radiation skin lesions with the mesenchymal stem cells (MSC) derived from human gingiva and their conditional medium concentrate (CCM) during animal studies. Material and methods: The study includes 80 white male Wistar rats weighing 210 ± 30 g at the age of 8–12 weeks, randomized into 4 groups (20 animals in each): control group (C), animal did not receive treatment; control with the introduction of the conditional medium concentrate (CCM) three times on days 1, 14 and 21; the introduction of MSC in a dose of 2 million cells per 1 kg three times on days 1, 14 and 21; the introduction of CCM in the estimated dose of 2 million cells per 1 kg three times on days 1, 14 and 21. Radiation burn simulation was performed (using on an X-ray unit at a dose of 110 Gy) and each animal was observed 17 times: at days 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105 and 112. Histological (stained with hematoxylin-eosin) and immunohistochemical (CD31, CD68, and VEGF) studies were performed. MSC was cultivated according to the standard procedure up to passages 3–5, the conditioned medium was collected and concentrated 10 times. The MSC immunophenotype (CD34, CD45, CD90, CD105, CD73, HLA-DR) and viability (7-ADD) were determined using flow cytometry. Results: Under the assessment of the animal skin on the day 7 in the CCM group, the area was significantly larger compared to the C, MSC, CM groups (р ≤ 0.05). In the CM group on the day 14 the area of the open wound surface and ulcers from day 28 to day 42 was significantly less, compared with the C, MSC and CCM groups (р ≤ 0.05). In group C, from 42 to 77 days of observation, an increase in the area of skin ulcers was observed compared with the CM and CCM groups (р ≤ 0.05). On the day 112, healing of skin ulcers in the CM group was observed in 40 %, in the MSC group in 60 %, and only in 20 % of animals in the CCM group, and in the C group it was not registered. Expression of VEGF marker on endothelial cells and stromal cells was observed in groups C and CM on day 28 and in groups MSCs and CCM on day 112. On the 28th day in the MSC group, the average number of vessels (CD31) in the field of view was 6.0, and on day 112 it was 12.75, р ≤ 0.05, in the CCM group – 19.10 and 28.6, respectively, р ≤ 0.05. An increase in the number of macrophages (CD68) was found in group C from 28 to 112 days (11.6 and 24.73, р ≤ 0.05), and in the CM group the decrease was 22.1 and 13.07, respectively, р ≤ 0.05. Conclusion: Thus, all used treatment modes of radiation skin lesions, including 3-fold administration of CM, MSC and CCM at a dose of 2 million cells per 1 kg, were effective and resulted in a reduction in the damage area, accelerated ulcer healing, and improvement of the regenerative processes. In addition, the use of MSCs led to the improvement of inflammatory processes’ vascularization and reduction in the radiation skin lesions.
Purpose: To study the effect of radiation sterilization at an ultra-high dose of 30 kGy on the cytocompatibility of decellularized vascular scaffolds repopulated with placenta MSCs. Materials and methods: The material of the study were aortas of laboratory animals (rabbits and rats, three vessels for each animal species), which were subjected to detergent - enzymatic perfusion decellularization by two protocols differing in reagents composition. Then the scaffold decellularized by the most efficient protocol was irradiated at a dose of 30 kGy and repopulated with placenta MSCs. As a control, the unirradiated matrix was seeded with cells of the same type. Histological staining of hematoxilin – eosin, IHC for type I collagen and Ki67, DAPI staining and quantitative assessment of genomic DNA were used to evaluate the effectiveness of decellularization and seeding. Scaffolds seeding was assessed by analyzing serial sections taken on day 1st, 3rd, and 4th of culture. Results: The scaffolds obtained in accordance with Protocol 1 were characterized by the absence of detectable cell nuclei, while the DNA content in them was significantly lower compared to Protocol 2. On the digitized images of sections of the unirradiated matrix, the cell nuclei were determined for routine H&E and DAPI staining while for the irradiated scaffold the cell nuclei were visualized on the border between the scaffold and fibrin gel only on DAPI stained section at 1st day of culture. The frequency of occurrence of Ki67+ nuclei on the 4th day of culture was significantly lower for the irradiated scaffold in comparison with the non-irradiated scaffold (7.5% and 29.8%, respectively, p=0.0054). Conclusion: Scaffold irradiation leads to loss of cytocompatibility of tissue-engineering constructs.
Purpose: The aim of the study was to study the effect of low X-ray doses on human mesenchymal stem cells (MSCs) in long-term cultivation in vitro. Material and methods: MSCs of the mucosal gum tissue of human were used. Cells were irradiated using an RUST-M1 X-ray unit (Russia) at doses of 50, 80, 100, 250 and 1000 mGy (dose rate40 mGy/min) and then cultivated according to standard methods. Immunological characteristics and viability of MSCs were evaluated on a FACSCanto II flow cytometer (Becton Dickinson CA, USA) for early and late passengers. Proliferative activity (PA) was evaluated using an xCelligence real-time cell analyzer (ACEA Biosciencs, Inc.). Results: It was shown that the proliferative activity (PA) of MSCs of the mucosal gum tissue which were irradiated at dose 50 mGy is comparable with the control group in long-term cultivation while doses of 100 and 250 mGy showed a decrease of PA. Also non-irradiated MSCs showed a significant decrease of the PA during cultivation in a conditioned medium from cells that received dose of 1000 mGy and an increase of PA during cultivation in a conditioned medium of cells that received doses of 50, 100 and 250 mGy. The cells were previously irradiated at dose 250 mGy showed adaptive response during cultivation in conditioned medium from cells that received dose of 1000 mGy. Сonclusion: The assessment of the effects of low radiation doses was focused on the bystander effect in the presented study. It was noted after adding conditioned media from irradiated cells to previously irradiated and non-irradiated MSCs. The bystander effects for low and high doses are different and their biological meaning requires further study. The phenomenon of adaptive response was shown after addition conditioned media from cells irradiated at dose 1000 mGy to pre-irradiated MSCs received a dose of 250 mGy. The obtained result leads to the conclusion that the effects of low doses can be positive. Thus, the results of study mainly correspond to the threshold nonlinear concept, according to which the effect is not proportional to the received radiation dose.
Introduction. Urethral stricture is a complex and urgent problem in operative urology. The main problem in the treatment of extended structures of the posterior urethra is the inability to form an adequate urethral site for augmentation urethroplasty with the common buccal graft, which has a priority in the treatment of penile strictures. The use of tissue acellular matrices may be promising in the development of reconstructive urology, which in the future will solve a number of problems associated with augmentation urethroplasty. The purpose of this article is to study the possibility of using a cell-free matrix of a donor artery as a free flat flap for stricture replacement urethroplasty on a model of laboratory animals (rabbits). Materials and methods. Donor blood vessels were Used, which were subjected to detergent-enzymatic perfusion decellularization. To assess the quality of the cell-free matrix, a histological study and an immunohistochemical study were performed. The cell-free flap of the donor artery was fixed to the protein envelope from the side of the simulated defect and posterior on-lay urethroplasty was performed.Results and discussion. The resulting matrix was characterized by the absence of detectable cell nuclei, preserved type I collagen, and a DNA content of no more than 50 ng / mg of tissue. In the postoperative period, normal motor activity of animals, normal urination, weight loss was not observed. The levels of C- reactive protein, creatinine, and urea in peripheral blood 5 months after surgery were within the normal range: 0.285±0.04839 mg / l, 93.5±8.057 mm / l, and 8.35±1.355 mm/l, respectively. If cystourethrography with the help of computer tomography data for stricture of the urethra is not revealed. During magnetic resonance imaging in the axial and sagittal projections, the patency of the urethra was indirectly confirmed. Conclusion. In a laboratory animal model, it was shown that the resulting cell-free flap has in vivo biocompatibility and can be used for replacement urethroplasty of posterior urethral strictures.
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