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: Study of the effect of paracrine factors, produced by MMSC of bone marrow during the cultivation, on the severity of local radiation injuries in the conditions of application in the early periods after irradiation. Material and methods: Experiments were performed on rats of the breed Wistar weighing 280 g. Rats were exposed locally in iliolumbar region of the back using X-ray machine LNC-268 (RAP 100-10) at a dose of 110 Gy (30 kV tube voltage, current 6.1 mA, filter Al 0.1 mm thick), dose rate is 21.4 Gy/min. Area of the irradiation field was 8.2–8.5 cm2. The conditioned medium obtained by culturing MMSC of rats’ bone marrow was administered in dose 1.0 ml (total protein 8 mg/ml) at 1, 3, 6, 8 and 10 days after irradiation. The severity of radiation damage to the skin and the effects of therapy were evaluated in dynamics by clinical manifestations, using planimetry and histological methods. Results: It was shown that in control animals and in rats, with the introduction of the conditioned medium, the values of the skin lesion area in the period up to the 29th day after irradiation practically did not differ, gradually decreasing in control animals from 5.9 ± 0.6 cm2 to 2.2 ± 0.3 cm2 at the 15th and 29th days after irradiation, respectively. Then, in the control group, the lesion area ranged from 1.4 ± 0.6 cm2 on the 50th day to 1.9 ± 0.8 cm2 on the 71st day. In the experimental group of animals, with the introduction of factors of the conditioning medium, a decrease in the area of the lesion and a stable dynamics of healing of radiation ulcers, beginning from the 36th day, there was a gradual decrease in the area of the lesion, which reached 0.2 ± 0.1 cm2 by the 71st day after irradiation. On the 64–71th day after irradiation, the difference between the areas of skin lesion in the experimental and control groups was statistically significant, p <0.05. The histological analysis showed that the use of paracrine factors obtained from MMSC in the process of cultivation significantly reduces the severity of the inflammatory reaction and accelerates the regeneration processes. Conclusion: Thus, the introduction of conditioned medium factors obtained during the cultivation of mesenchymal stem cells of the bone marrow facilitates a more easy flow of the pathological process and the healing of radiation ulcers after local radiation damage to the skin of rats. Apparently, the favorable effect of paracrine factors introduced in the early periods after irradiation, with severe local radiation injuries, is associated with their effect on pathological processes in the inflammatory-destructive stage.
Medical examinations for employees of organizations using sources of ionizing radiation, have a number of features, including the need for a «specialized» medical examination with mandatory psychophysiological examination.
Purpose: Analysis of the prevalence of diseases of non-professional genesis in individuals with intake of radionuclides through damaged skin and served by health care institutions of the Federal Medical and Biological Agency of Russia. Material and methods: The database of the “Industry register of persons with occupational diseases” includes the health indicators of individuals with intake of radionuclides through damaged skin and a population of 185 patients aged 68.9 ± 1.0 years for 2014 (166 men aged 68.3 ± 1.0 years and 19 women aged 74.0 ± 3.4 years). The coding of diseases of non-professional genesis was carried out according to the International Statistical Classification of Diseases and Problems Related to Health (ICD-10). Student’s criterion was used as a criterion of statistical validity (p < 0.05). To assess the health of persons counted in the register, the prevalence rate of diseases of non-professional genesis, calculated for 1000 patients with occupational diseases, was used. An intensive indicator error was calculated ( ± m 95 %). Results: The prevalence of non-occupational diseases is 1637.8 ± 94.1 (men 1614.5 ± 98.6, women 1842.1 ± 311.4; p > 0.05). Disorders of the musculoskeletal system occupy the first place – 340.5 ± 34.8; 20.8 % (men 313.3 ± 36.0; 19.4 %, women 578.9 ± 174.6; 31.4 %; p > 0.05). Of these, dorsopathies accounted for 286.5 ± 33.1; 84.1 % (men 265.1 ± 34.3; 84.6 %, women 473.7 ± 114.6; 81.8 %; p > 0.05). Diseases of the digestive organs are 270.3 ± 32.7; 16.5 %. The highest rates in persons with diseases of the esophagus, stomach and duodenum 205.4 ± 29.7; 76.0 % (men 210.8 ± 31.7; 76.1 %, women 157.9 ± 83.7; 75.0 %; p > 0.05). The third ranking place is divided by diseases of the eye and its adnexa 200.0 ± 29.4; 12.2 % (men 192.8 ± 30.6; 11.9 %, women, 263.2 ± 101.0; 14.3 %; p > 0.05) and circulatory system diseases 200.0 ± 29,4; 12.2 % (men 174.7 ± 29.5; 10.8 %, women 421.1 ± 113.3; 22.9 %; p > 0.05). In the structure of diseases of non-professional genesis, the diseases of the first four ranking places, including respiratory diseases, make up 73.6 % (men 72.4 %, women 82.9 %). Conclusion: Among the diseases in persons with the intake of radionuclides through damaged skin are the most common disorders of the musculoskeletal system, diseases of the digestive system, diseases of the eye and its appendage apparatus. In terms of frequency, they are inferior to diseases of the circulatory system and respiratory organs. As one of the solutions to the problem of registration in persons with signs of contamination of radionuclides of damaged skin in the framework of ICD-10, it is proposed to introduce code S61.2 as “Open wounds of wrist and hand with intake of radionuclides”.
Purpose: To analyze the features of the clinical course of chronic radiation syndrome (CRS) due to external non-uniform chronic exposure to prolonged household contact with a lost source of ionizing radiation. Material and methods: Analysis of 2 clinical observations of patients who developed subacutecourse of CRS and chronic radiation dermatitis due to external non-uniform exposure of the lost sources of ionizing radiation are presented. Results: Boy A. K. from the age of 1 year for 7 years was external radiation exposed (for 1.5 years non-uniform exposure ) to the total dose according EPR tooth enamel about 6.3 Gy, according to the data of retrospective dose recovery on red bone marrow using voxel modeling – 26 (19–37) Gy. F. V. V., male, 38 years, was external non-uniform radiation exposed for 5 months, the total dose according cytogenetic studies of 7.9 Gy (dose rate about 0.035 Gy/h). During the examination in the hospital, the patients were diagnosed with CRS. Within the framework of the bone marrow syndrome, deep thrombocytopenia, moderate leuko- and neutropenia, and moderate anemic syndrome were observed. The latter is not typical for the typical course of CRS and is a criterion indicating a subacute course of the disease. In addition, signs of chronic radiation dermatitis were found in the projection of the action of the ionizing radiation beam. After stopping the radiation exposure, the patients did not recover their hematopoietic function, and in the period of immediate consequences, they developed myelodysplastic syndrome (MDS) with further transformation into acute leukemia. Conclusion: 1. Accidental prolonged household or criminal contact with a source of ionizing radiation can lead to the formation of CRS with an atypical subacute course and the formation of MDS with transformation to acute leukemia in the outcome of the disease or in the period of its consequences. 2. It can be assumed that with external non-uniform radiation exposure, leading to the development of CRS and chronic radiation damage to the skin, agranulocytosis in the subacute course of CRS may be absent. 3. Adverse prognostic signs for the development of MDS and leukemia in the outcome or in the period of the consequences of subacute CRS with non-uniform exposure are long-lasting deep thrombocytopenia and anemic syndrome after the end of radiation exposure.
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.
Treatment of local radiation injuries (LRI) is one of challenges of radiation medicine, at the same time tactics and strategy of therapy of these pathologies remain up to the extremity not developed. Recently the increasing place in a clinical practice is borrowed with various methods of cellular and tissue therapy. The certain successes in development and clinical application of the various biomaterials stimulating reparative processes and replacing defects of a skin are reached. The epidermis is one of the important functional units of a skin as organ, and its ability to self-renewal is defined by presence stem cells (Terskih V.V. et al., 2001). As a result of damage which can wear chemical, mechanical, thermal or radiative character, the structure of a epidermis and a derma varies, structurally functional units disappear, and keratinocytes and fibroblasts get a new phenotype. However, not looking at huge quantity of works of the proliferative abilities devoted to studying keratinocytes and fibroblasts and as to their use with the purpose of replaceable therapy at influence of various injuries factors, experience of clinical application of these technologies at patients with LRI for today is absent. We for the first time used a method of replacement of defect of a skin with use autological keratinocytes and fibroblasts at the patient with LRI. With the purpose of application of cellular technologies to the patient the intraoperative biopsy of a healthy dermal graft is made. In the further it was carried out growth autological keratinocytes and fibroblasts. Creation of a design includes 4 stages: a biopsy of a donor material; growth cells in vitro; creation tissue engineering designs; transplantation tissue engineering designs in area of injury. Use of cell technologies alongside with traditional surgical and therapeutic methods of treatment LRI shows high efficiency and perspectivity of this direction. Thus, studying of specificity of radiative influences on cells of a skin and as use autologic keratinocytes and fibroblasts in the program of treatment of patients with local radiation injuries for replacement of defects, represents appreciable scientific and practical interest, and their application with the purpose of optimization of current reparative processes has a pathogenetic substantiation.
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