Purpose. To compare the experimental results of corneal repair and regeneration after transplantation of combined bioconstruction with cultivated or non-cultivated (minimally manipulated) buccal epithelial cells in total limbal stem sell deficiency. Material and methods. The study was conducted on 24 eyes of Gray Chinchilla rabbits. At the first stage rabbits underwent bilateral total limbectomy and mechanical de-epithelialization of the cornea, resulted in fibrovascular pannus development. After superficial keratectomy to transparent layers the combined bioconstructions with autologous cultivated epithelial buccal cells were placed on the corneas of group 1 (12 eyes), bioconstruction with non-cultivated (minimally manipulated) buccal epithelial cells were placed on corneas of group 2 (12 eyes). Temporary tarsorrhaphy was performed for 5 days. In the postoperative period the area of the de-epithelized surface, neovascularization and corneal transparency were evaluated. Results.In both groups we noted reduction of corneal erosion at all periods. The dynamics of recovery processes was equal, the difference between the groups was statistically significant only at 15th and 60th days (p = 0.028 and p = 0.026). Groups did not differ in neovascularization severity and corneal transparency (p > 0.05). Conclusion.Due to the absence of obvious differences in cornea restoration, the use of non-cultivated epithelial cells of the oral mucosa seems to be a promising treatment method in treatment of total limbal stem sell deficiency. For wide application in ophthalmological practice, further study of their effectiveness in clinical trials is required. Keywords: limb, limbal stem cell deficiency, epithelium of the oral mucosa, cornea, rabbits

Background: Controlled generation of active forms of oxygen and nitrogen stimulates wound healing processes. One of the sources of controlled generation of active forms of oxygen and nitrogen is low-temperature argon plasma. Low-temperature plasma activates various forms of cellular activity. However, the subtle mechanisms of such stimulation remain unclear. Thus, there is a need for a detailed study of the biological effect of low-temperature argon plasma on human cells.Objective: to evaluate the effect of low-temperature argon plasma on the structure and proliferative activity of human fibroblasts in vitro.Materials and methods. Human fibroblast culture (line M-21, passage 25, Chumakov Institute of Poliomyelitis and Viral Encephalitis) was used in the work. After exposure to low-temperature argon plasma, the cells were cultured for 3 days in DMEM/F12 medium supplemented with 10% Gibco bovine embryo serum, then morphofunctional analysis of the cells was performed using vital staining. After 3 days, the cells were passaged (reseeded in other Petri dishes with a 1:2 dilution), then the cells were cultured for 3 days, and a repeated morphofunctional analysis was performed. All work with cell cultures was carried out in accordance with the international GMP standard.Results. Stimulation of the proliferative activity of fibroblasts was noted in the second passage after treatment with low-temperature argon plasma from a distance of 10 cm for 30 and 45 seconds and 15 cm for 45 seconds.The use of a collagen bandage screen significantly increases cell survival when exposed to plasma from a distance of 10 cm for 15 seconds. With screening, the decrease in the integrity of cell membranes after plasma exposure is less pronounced than with exposure without screening. When reseeding, cells treated in the presence of a collagen screen exhibit high proliferative activity and restore membrane integrity.

Experiments on 39 male Wistar rats were performed. A metal-organic coordination polymer modified with iron oxide and ascorbic acid (composite) was administered intravenously to healthy rats at doses of 25 and 50 mg/kg. The anti/prooxidant activity of the composite was assessed 3 and 24 hours after administration. The composite in both doses was shown to have no significant effect on the homeostasis of prooxidants/antioxidants in the blood serum in healthy animals. In high doses, the composite enhances apoptosis of lymphocytes, which indicates its possible use as a catalyst for free radical processes for further use in biomedicine.

We studied morphofunctional features of human platelets exposed in vitro to low-pulse laser radiation (LPLR) with different wavelengths. Platelet-rich plasma (PRP) was irradiated at wavelengths of 635, 488, and 355 nm. LPLR with λ=635 nm and 1 W power after 10 min caused degranulation of many platelets, formation of small platelet conglomerates, and accelerated platelet adhesion on glass. LPLR with λ=635 nm and 2 mW and LPLR with λ=488 nm did not cause significant changes in the morphofunctional platelet status. LPLR with λ=355 nm induced massive platelets’ deformation, sharp decrease in their morphofunctional status without activation.

Purpose. To estimate the effectiveness of using of platelet-rich plasma lysate (PRP lysate) for prevention and treatment keratolysis in patients after urgent keratoplasty. Material and methods. To compare the effectiveness of treatment with PRP lysate after urgent keratoplasty we formed the comparison group and the study group (20 people in total). Each group included 10 patients: 5 with corneal perforation and 5 with graft lysis. After urgent keratoplasty all patients received standard treatment. 10 patients from the study group received platelet-rich plasma lysate in subconjunctival injections and instillations. The assessment of the condition of the cornea was carried out using clinical and instrumental studies, including visometry, biomicroscopy with fluorescein staining, photoregistration and OCT on the Heidelberg Engineering “OCT Spectralis” apparatus. Results.Treatment with PRP lysate in all patients of the study group led to 100 % epithelization of the graft up to 7 days after surgery. The average area of graft epithelization on day 7 was 55,9 ± 12,9 % in patients before the first keratoplasty, and 38,22 ± 14,5 % in patients after rekeratoplasty (p < 0,01 compared with the study group. In all patients of the study group, the graft was preserved. In the comparison group, 70 % of patients had keratolysis in the long term after surgery. Conclusions. The use of PRP lysate after urgent keratoplasty may be a promising method for preventing and treating keratolysis. Keywords: keratoplasty, matrix metalloproteinases, tissue inhibitor of matrix metalloproteinases, platelet-rich plasma

Introduction. Harvesting of pooled platelet concentrates (PC) significantly expands the volume of transfusion support and could be used to create strategic platelet reserve.The aim of work. To compare the quality of pooled and apheresis platelet concentrates.Materials and methods. We studied platelet fractions (PF), isolated from the whole blood of cadre donors with Reveos system, platelet concentrates, obtained by pooling PF (Pooled PC), and platelet concentrates harvested by automatic apheresis. In all cases 2 days passed from the moment of blood donation to the platelet examination. In PC samples we studied cellular composition, morphofunctional parameters of platelets, pH and electrochemical parameters, hypotonic shock response.Results. The morphofunctional platelet rate in PF was significantly reduced, comparing to the normal levels. At the same time, the morphofunctional parameters of platelets did not significantly differ in PF and pooled PC. The pH values, the total content of platelets and leukocytes in the pooled PC corresponded to the requirements of The rules for procurement, storage, transportation and clinical use of donated blood and its components (2019). The total platelet count, the level of annexin-positive platelets, did not significantly differ in apheresis PC and pooled PC, hypotonic shock response was correct in apheresis PC and pooled PC. On the other hand, the level of platelets with granules in pooled PC was 1,8 times lower than in apheresis PC, other morphofunctional parameters of platelets in pooled PC were also reduced. Pooled PC were characterized by more positive level of open circuit potential and lower antioxidant capacity compared with apheresis PC on the same storage time.Conclusions. Platelet pooling with Reveos system is acceptable for obtaining PC doses that comply with Technical Regulations. Nevertheless, methods of pooled PC harvesting need further optimization.

Allogeneic tendon grafts are seriously demand in knee joint plastic surgery. The novel method of tendon cryopreservation, including sterilization with supercritical carbon dioxide, was developed in N.V. Sklifosovsky Research Institute for Emergency Cryopreserved tendons retain their normal fiber structure without significant loss of mechanical properties. At the next stage it was necessary to evaluate cryopreserved tendons′ integration inside bone canal in experimental animals. The aim of study. To evaluate morphologic changes of autologous and allogeneic tendons inside the femur in rats and to determine the effect of tendon transplantation on the physical activity. Material and methods. The study was conducted on white inbreed male rats. Three groups of animals were formed: the control group (animals without tendon transplantation), the 1st experimental group – animals with autologous tendon transplantation, the 2nd experimental group – animals with allogeneic tendon transplantation. In animals of the experimental groups the through channel was formed in the distal metaepiphysis of the femur and a tail tendon graft 0.5 x 0.1 cm was placed there. To assess the physical activity of the animals, we studied maximum distance that the animals could run 3 and 6 weeks after transplantation was determined, using treadmill test. The graft structure was evaluated on histological preparations in transmitted light, stained with hematoxylin-eosin and Van Gieson′s stain. To assess the preservation of collagen fibers we checked the autofluorescence intensity of collagen. Results. According to the treadmill test, the distance run by the animals of both experimental groups did not significantly differ from the values in the control group. Histological analysis after 3 weeks in both experimental groups revealed signs of fibers′ decomposition in the absence of inflammatory infiltration and maintaining close contact with bone trabeculae. The autofluorescence intensity of the collagen fibers in grafts corresponded to normal or was close to normal. After 6 weeks, the animals of both experimental groups revealed areas of graft fusion with their own bone, Sharpe fibers were actively formed. In both groups, numerous small vessels with diameters up to 10 microns were detected in the area of tendon-bone contact. Infiltration of grafts by inflammatory cells was absent or very insignificant, active migration of fibroblasts to the tendon area was also not observed. In both groups, tendon grafts had areas where fiber decompactization was observed. In the area of contact with the bone, the autofluorescence of tendon fibers was sharply increased, which indicates the chemical cleavage of collagen. At 3 and 6 weeks after transplantation the effect of fixation (integration) of the tendon with bone tissue was observed in both experimental groups. Conclusions. Allogeneic tendon grafts did not cause a pronounced inflammatory or immune reaction in experimental animals. 6 weeks after transplantation of autologous and allogeneic tendons, the integration of grafts inside the femoral canal was observed. Cryopreserved allogeneic tendons were able to integrate into the body's own tissues without pronounced structural and functional disorders. According to the treadmill test, the distance covered by the animals of both experimental groups did not differ statistically significantly from the values in the control group (without tendon transplantation) after 3 and 6 weeks

Purpose: to evaluate the content of tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1) in platelet preparations obtained in different methods.Material and methods. Platelet-rich plasma (PRP), platelet-poor plasma, and a suspension of platelets washed from plasma (WP) were isolated from the blood of 10 volunteer donors, and a morphofunctional analysis of platelets was performed. After that, a platelet lysate was prepared from PRP, WP, and platelet-poor plasma. The concentration of TIMP-1 in the lysates was determined using enzyme immunoassay.Results. The level of TIMP-1 in the lysates of PRP and WP did not differ significantly; in platelet-poor plasma, the level of TIMP-1 was significantly lower than in the lysates of PRP and WP (p = 0.003). In all types of preparations, the concentration of TIMP-1 was 2-4 times higher than similar values in the blood serum of healthy people, as reported in other studies. A weak correlation was found between the TIMP-1 concentration and the platelet number in PRP, and there was no correlation between the content of platelets with granules and the TIMP-1 level in the lysates. The presence of leukocytes in the initial PRP also did not affect the TIMP level in the final lysates. A strong correlation was found between the TIMP-1 concentration in the WP lysate and in platelet-poor plasma, as well as a direct correlation between the total platelet content and the TIMP-1 level in the PRP lysate.Conclusions. Preparations with high concentrations of TIMP-1 can be obtained both from concentrated platelet suspensions and from platelet-poor plasma. To optimize the method, it is necessary to study the effect of platelet preparations with TIMP on various biological models.

A comprehensive study of hemocompatibility and gas permeability of 1,2-disubstituted polyacetylenes, namely poly(1-trimethylsilyl-1-propyne) and poly(4-methyl-2-pentyne), was carried out. The polymers were synthesized started from 1-trimethylsilyl-1-propyne and 4-methyl-2-pentynemonomers on the catalytic systems NbCl5 and NbCl5/n-Bu4Sn to form homopolymers containing 50 and 55

Morphofunctional characteristics of human platelets in the presence of 0.1-5 mM ascorbic acid were studied. The platelet ability to form lamellae and the preservation of granules in platelets in suspension and during adhesion were evaluated. Ascorbic acid in concentrations of 0.1-1 mM induced no visible changes in platelet structure and did not affect their adhesion activity, but suppressed lamella growth and degranulation in adherent platelets in a dose-dependent manner. The maximum preservation of granules was revealed in the presence of 0.5 mM ascorbic acid (55% in 1 h from the moment of adhesion). In the presence of 2-5 mM ascorbic acid, spontaneous activation and degranulation of platelets was observed. Thus, ascorbic acid is capable of both suppressing and stimulating platelet activity. In concentrations of 0.5-1 mM ascorbic acid can be used to stabilize granules in adherent platelets.

Introduction . Biological grafts, including bone chips and collagen, are supposed to be promising in the treatment of bone tissue treatment. Nevertheless, manufacturing of these grafts still needs to be standardized. Aim . To optimize methodology of osteoplastic material production, based on allogenic 1 type collagen and bone chips. Material and methods . Osteoplastic material grafts were produced, using with allogeneic bone chips 180-800 µm and type 1 collagen solution in acidic acid. We studied total integrity of graft, collagen quality, morphofunctional properties of line M-22 human cells interacting with different type of osteoplastic material grafts. Results . Procedures for manufacturing the osteoplastic material did not significantly affect the quality of collagen in its composition, while lyophilized grafts had pronounced acidogenic and toxic effects in cell culture. Soaking osteoplastic material in isotonic solution for 30 min or longer increased its biocompatibility in vitro. Adhesion properties of osteoplastic material widely varied depending on collagen concentration and bone chips size. Conclusion . Osteoplastic material prepared with allogeneic bone chips of 180–800 µm and collagen solution in acidic acid had pronounced acidogenic and toxic effects in vitro that could be considerably reduced by soaking in isotonic solution. Varying the collagen concentration and the size of bone chips one could produce biocompatible osteoplastic material grafts with high and low adhesion properties.

Тяжесть течения новой коронавирусной инфекции (COVID-19), вызванной вирусом SARS-CoV-2, во многом обусловлена развитием у больных иммунопатологического состояния. У 85% пациентов с тяжелым течением COVID-19 отмечается лимфопения, развитие которой может быть связано с повышенным апоптозом лимфоцитов. ЦЕЛЬ ИССЛЕДОВАНИЯ Провести анализ апоптотической гибели лимфоцитов и изменений белков — регуляторов программируемой смерти клеток у больных с тяжелым течением COVID-19. МАТЕРИАЛ И МЕТОДЫ Обследованы 93 пациента, находившихся на лечении в реанимационных отделениях ГБУЗ «НИИ СП им. Н.В. Склифосовского ДЗМ». Всех обследованных пациентов в зависимости от степени тяжести и исхода заболевания распределили в три группы: 1-ю группу составили 53 пациента с благоприятным течением и исходом заболевания, во 2-ю группу включили 26 пациентов с неблагоприятным течением и благоприятным исходом заболевания, в 3-ю группу вошли 14 пациентов с неблагоприятным течением и исходом заболевания. Забор крови для исследования маркеров апоптоза проводили на 5—12-е и на 14—18-е сутки с момента начала заболевания. Количественные параметры апоптоза лимфоцитов крови оценивали с помощью проточной цитометрии. Регуляторные белки апоптоза (фосфорилированную АКТ, JNK, BAD, Bcl-2, p-53, активную каспазу 8 и 9) определяли на платформе Luminex, также оценивали концентрацию лейкоцитов, относительное и абсолютное содержание лимфоцитов, концентрацию С-реактивного белка, прокальцитонина, лактатдегидрогеназы. РЕЗУЛЬТАТЫ Группы статистически значимо различались по тяжести состояния по шкале NEWS (p=0,001) и органной дисфункции по шкале SOFA (p=0,001), а также по уровню С-реактивного белка (p=0,001), выраженности лимфопении (p=0,001), содержанию CD14+HLA-DR+-моноцитов (p=0,001). Количественные параметры апоптоза лимфоцитов не коррелировали с развитием лимфопении. Наиболее высокие показатели апоптоза лимфоцитов отмечены у пациентов с благоприятным течением и исходом заболевания. Не выявлена корреляционная зависимость концентрации лимфоцитов в венозной крови от уровня белков — регуляторов апоптоза. ЗАКЛЮЧЕНИЕ У больных с тяжелым течением COVID-19 в ответ на вирусную агрессию отмечено нарушение индукции апоптоза лимфоцитов как через внешний, так и через внутренний путь активации. У умерших больных преобладало влияние проапоптотических факторов при снижении активности антиапоптотических факторов.

Structural and functional properties of the human tendon tissue complicate process of tendon-based grafts preserving. In the process of preservation, it is necessary to maintain mechanical and functional parameters of the tendon tissue as well as to provide sterility and biological safety of the graft. One could conclude effectiveness of techniques, combining tendon conservation at low temperatures and sterilization with the supercritical carbon dioxide. Aim of work : to find optimal conditions for allogeneic tendon grafts sterilization with supercritical carbon dioxide. Materials and methods . Allogeneic tendons were prepared from the tissue donors, following the rules of asepsis and antisepsis. After quarantine and confirmation of the absence of blood-borne infections the tendon grafts were divided into 3 groups: in the control group the tendons were not subjected to cryopreservation and sterilization procedures; in 2 experimental groups tendons were treated with a cryoprotector 10 % dimethyl sulfoxide (DMSO) solution and sterilized with supercritical carbon dioxide with slow gas discharge (group 2) or with fast gas discharge (group 3); the duration of sterilization in both groups varied from 1 to 12 hours. The toxicity of the grafts was assessed in human mesenchymal multipotent stromal cells (MMSC) culture on the 3rd and 7th days. The sterility of the grafts was confirmed on the 7th and 14th day by bacteriological culture on thioglycol medium and Saburo broth. Mechanical tests were performed on testing machine LLOYD Instruments LR5K Plus with the tensile speed 5 mm/min. Results. Histological analysis showed, that in the group with slow gas discharge collagen fibers retained their integrity and topography and contained only local minor tears; in all terms of treatment, the tendon grafts were sterile and nontoxic. On the contrary, in the group with fast gas discharge there was marked damage of the collagen fibers and growth of bacterial and fungal flora was observed in the culture. The grafts of the control group and the group, where sterilization with supercritical carbon dioxide was performed with slow gas discharge for 1-12 hours, were used to evaluate mechanical characteristics. In the preserved tendons the stiffness and ultimate strain did not significantly differ from the similar values in the control (p > 0.05), on the contrary, the level of ultimate strain in all experimental specimens was significantly reduced by 1.5-2.1 times comparing to the control. Young's modulus and load at rupture in the control grafts and grafts, sterilized for 1 hour, had similar values, whereas in grafts, sterilized from 3 to 12 hours, these parameters were 1.4-2.1 times lower (p < 0.05). Conclusions. The suggested technique of tendon preservation, using cryoprotector DMSO and sterilization with the supercritical carbon dioxide, allows to obtain sterile and non-toxic grafts. The structure of cells and fibers of the preserved tendons did not significantly disturbed. For supercritical carbon dioxide sterilization of tendon grafts the most optimal processing time is 1 hour.

Aim of study. To evaluate the effectiveness of allogeneic tendon, sterilized with supercritical carbon dioxide, in restoration of the anterior cruciate ligament (ACL) during clinical example. Material and methods. The patient was admitted at the N.V. Sklifosovsky Research Institute for Emergency Medicine with the diagnosis: an old rupture of the anterior cruciate ligament of the right knee joint, rupture of the internal meniscus, chondromalacia of the inner condyle of the right femur 2-3 art., according to the ICRS classification. From the anamnesis, it became known about the previous 2 operations for ruptures of the ligaments of the right knee joint and the use of autologous tendons as a plastic material. A conversation was held with the patient about all possible methods of ACL repair and a choice was made in favor of using a cryopreserved allogeneic tibialis anterior tendon graft as a graft due to a shortage of autologous tissues. Allogeneic tendons were taken from tissue donors, treated with 10% dimethyl sulfoxide, sterilized with supercritical carbon dioxide and frozen at -80 degrees C. After confirming the absence of toxicity, as well as the results of bacteriological examination, the allogeneic tendon graft was used for ACL repair. Results. The early postoperative period was uneventful. On the 12th day postoperative wounds healed by primary intention. An MRI examination revealed a graft of the anterior cruciate ligament in the formed bone canals; the graft was not thickened and had a homogeneous structure. After 28 days, the patient gradually began active and passive development of movements in the knee joint. After 6 weeks the range of motion and strength in the right knee joint was comparable to the left knee joint. After 4 months, according to magnetic resonance imaging, ligamentation of the allogenic graft and "synovial sleeve" formation were observed. Conclusion. According to the clinical course and time of rehabilitation, the technique of using allogeneic tendons during ACL plastic surgery was comparable to the use of an autologous graft. The clinical experience of using allogeneic tendons at the early postoperative stage could be assessed as successful. Further observation is necessary to fully assess the reconstruction and integration of the tendon.

Purpose : to compare the effectiveness of conventional preserved amnion and amnion saturated with platelet-rich (PRP) plasma lysate for the treatment of persistent corneal erosions (PCE) after high-risk keratoplasty. Materials and methods . 40 patients with persistent corneal erosions after high-risk penetrating keratoplasty, followed up for 12 months, were divided into two clinical groups of 20 people each. The main group of patients, aged 34 to 84, received Flexamer amniotic membrane + PRP lysate, while the comparison group, aged 41 to 80, received Flexamer amniotic membrane only. Amniotic membrane coating was used in persistent corneal erosions of penetrating keratoplasty after unsuccessful conservative treatment. The amnion was sewn 2 mm from the limb with a continuous suture and covered with a soft contact lens. As a source of platelets, we used the blood of healthy volunteers, from which platelet-rich plasma with platelet concentration of over 1000 thousand/µl was taken, which was then frozen at -40 °C and defrosted at 0…4 °C to obtain PRP lysate. The criteria for evaluating the effectiveness of both groups were the times of epithelialization, the number of amniotic membrane coatings, and the number of preserved transplants at the end of the follow-up. Results . Lyophilized amniotic membrane saturated with autologous PRP lysate growth factors was shown to be biocompatible. It was found to be safe for the patients, to reduce the epithelialization time, to reduce the number of operations required to cover the PÑE with the amnion, and to increase the likelihood of successful transparent and translucent engraftment of a penetrating keratograft. Conclusion . The use of a lyophilized amniotic membrane enriched with growth factors of autologous PRP lysate is a promising method for the treatment of PCE of the penetrating corneal graft after high-risk keratoplasty.

Purpose: To study the biological effect of a combination of platelet lysate and amniotic membrane, preserved by various techniques, on human buccal epithelium culture. Materials and methods. Human amnion transplants were preserved using 3 methods: silicate drying, lyophilization, cryopreservation. The blood of healthy volunteers was used as a source of platelets. Platelet-rich plasma (PRP) with a platelet content over 1000 thousand/mcl and more was isolated from the donors blood, frozen at -80 °С and defrosted at 0–4 °С to prepare platelet lysate. Growth-stimulating effect of the amnion transplants was studied in different groups: control group 1 — without amnion and without PRP lysate; control group 2 — PRP lysate without amnion; experimental group 1 — amnion without PRP lysate; experimental group 2 — amnion samples combined with PRP lysate. The study was carried out on the example of human buccal epithelium culture of 3–5 passages. The dynamics of cell growth was evaluated after 1, 2 and 3 days from the moment of seeding. The number of cells and their viability were evaluated using original methods based on vital cell staining and their examination in a fluorescent microscope. Results. All samples of preserved amnions were non-toxic and did not damage the structural and functional characteristics of the buccal epithelium. On the other hand, the use of amnion without PRP lysate did not have a growth-stimulating effect on cells. Among the amnion samples combined with PRP lysate, the combination of lyophilized amnion and PRP lysate was the most effective during the entire study period. Conclusions. Silicate drying, lyophilization and cryopreservation of the amniotic membrane makes it possible to obtain biocompatible and non-toxic transplants, based on human amnion. Lyophilized amnions are the most optimal for saturating PRP lysate. The combination of lyophilized amnion and PRP lysate stimulates cell growth in vitro without violating their structural integrity.

Introduction. Tendon grafts are widely demanded in reconstructive plastic surgery. Allogeneic tendons potentially have a number of advantages. However, the method of long-term storage of allogeneic tendons has not been optimized to date.Aim. Selection of an optimal cryoprotectant for the storage of human tendon allografts at ultra-low temperatures, which allows preserving the native tissue structure.Material and methods. We studied M. tibialis anterior tendon grafts taken from tissue donors. Endocellular/penetrating cryoprotectants (dimethyl sulfoxide, polyethylene glycol-400, glycerol) and exocellular/non-penetrating cryoprotectants (glucose solution, albumin solution) were used in the cryopreservation process. Tendon mechanical properties were evaluated using rupture and stretching-shear test, the general morphology of tendons, topography, dense and integrity of collagen fibers, preservation of cellular elements were microscopically evaluated.Results. Histological analysis showed that the safety of collagen and elastin fibers differed depending on the cryoprotectant used. At the same time, micro-fractures of collagen fibers were microscopically detected in all the experiments. In the presence of dimethyl sulfoxide, polyethylene glycol-400 and their combination the structure of collagen fibers and cells did not undergo visible changes compared to the control, whereas in all the experiments with non-penetrating cryoprotectants the topography and orientation of the fibers were clearly disturbed, deformation of many cells in the tendons was also observed.Conclusions. Cryoprotectants based on dimethyl sulfoxide, polyethylene glycol and their combinations allowed us to preserve the structural integrity of allogeneic tendons. Non-penetrating cryoprotectants did not effectively preserve the integrity of collagen fibers and cells in the tendons and cannot be recommended for cryopreservation.

We studied the growth-stimulating effect of platelet-based preparations obtained by different methods on cultured human fibroblasts M-22. Platelet lysates prepared from platelet-rich plasma, platelet-poor plasma, concentrated suspension of platelets washed from plasma, and platelet-rich plasma activated with calcium chloride (ActPRP) were used. The volume of the platelet preparations was 10-500 μl per 104 cells. The most effective dose of platelet-rich and platelet-poor plasma in cell culture was 20 μl, whereas for ActPRP, the most effective dose was 500 μl. Lysates of platelet-rich and platelet-poor plasma in doses of 100-500 μl inhibited fibroblast growth and disturbed their structural integrity. At the same time, lysates of washed platelets in doses of 10-500 μl stimulated cell growth and preserved their viability. An inverse correlation was found between the number of cells in culture and the level of proinflammatory cytokines in the platelet preparations.

Введение. При поверхностных ожогах процесс репарации происходит благодаря активности стволовых клеток и клеток-предшественников, сохранившихся в ране. Стимуляция миграционной, пролиферативной и секреторной активности этих клеток может быть инициирована с помощью компонентов, содержащихся в гранулах тромбоцитов. Значительно повысить сохранность тромбоцитарных гранул можно путем предварительной стабилизации тромбоцитов с помощью наночастиц серебра. Цель исследования - изучение репаративного эффекта коллагеновых повязок, насыщенных тромбоцитами, при лечении мышей с поверхностными ожогами. Методика. В работе использовались 3 типа раневых покрытий: коллагеновая повязка без тромбоцитов (контроль); повязка с нативными тромбоцитами (1-я опытная группа); повязка с тромбоцитами, предварительно стабилизированными 2,5 мкМ наносеребра (2 -я опытная группа). Тромбоциты выделяли из венозной крови доноров-добровольцев, консервированной на ЭДТА. Для стабилизации тромбоцитов в исходную суспензию с тромбоцитами предварительно добавляли раствор наносеребра до достижения концентрации 2,5 мкМ и инкубировали при 22 °С в течение 1 ч. Во всех опытах общее количество тромбоцитов с гранулами (биологически полноценные тромбоциты) в повязках составило 30-31 млн. После нанесения тромбоцитов раневые покрытия экспонировали в течение 30 мин при 37 °С, затем удаляли всю жидкую фракцию с неадгезировавшими тромбоцитами. Готовые повязки хранили при -40 °С и размораживали непосредственно перед экспериментом. Результаты. Через 3 сут у животных контрольной группы наблюдалась выраженная воспалительная реакция и значительная деформация коллагеновых волокон дермы, в опытных группах эти процессы были менее выражены. Использование коллагеновых повязок с тромбоцитами существенно усиливало миграцию эпителиальных клеток из дериватов кожи, а также миграцию фибробластов из глубоких слоев дермы. Через 5 сут эпителизация раны в контроле была частичной, тогда как у опытных животных эпителий был непрерывным по всей площади раны. В опытных группах сохранялась высокая миграционная активность эпителиальных клеток и фибробластов. Структурно деформированные волокна коллагена не выявлялись в опытных группах. Декомпактизация и отечность волокон в контрольной группе наблюдалась по всей глубине дермы, тогда как в опыте отечные волокна присутствовали только в сетчатом слое дермы. Заключение. Использование коллагеновых повязок с тромбоцитами ускоряло процессы эпителизации и восстановления волокон дермы на фоне сохранения локальной инфильтрация раны воспалительными клетками. Activity of stem cells and primordial cells is the main reparative factor in healing of superficial burns. Components of platelet granules could stimulate cell migration, proliferation, and secretion. Survival of platelet granules can be enhanced by prior platelet stabilization with silver nanoparticles. The aim of this work was to study reparative effect of collagen bands, saturated with platelets, on mice with superficial burns. Methods. Three types of wound coatings were used: 1) collagen bands without platelets (control); 2) collagen bands with native platelets (1st experimental group); 3) collagen bands with platelets, prestabilized with 2.5 µM nanosilver (2nd experimental group). Platelets were harvested from venous blood of volunteer donors preserved in EDTA. Platelet were stabilized by incubation with 2.5 µM nanosilver at 22°С for 1 hr. In all experiments, the collagen bands contained 30-31 million platelets with granules, i.e., biologically normal platelets. Collagen bands were incubated with platelets at 37°С for 30 min, and then all solution with unadhered platelets was eliminated. Experimental bands were stored at -40°С and defrosted immediately before experimental treatment. Results. After three days of treatment, the control group had a pronounced inflammatory reaction and significant deformation of dermal collagen fibers. In the experimental groups, these processes were less pronounced. Collagen bands with platelets significantly increased migration of epithelial cells from the skin derivatives and migration of fibroblasts from the deep dermal layers. After five days, the epithelization of wounds in the control group was partial, while in experimental groups, epithelium covered all areas of the wound. Experimental groups maintained high migration activity of epithelial cells and fibroblasts. Structurally deformed collagen fibers were detected sporadically in the control group and were not detected in the experimental groups. Decompactization and swelling of fibers in the control group were observed throughout the depth of the derma, while in the experiment groups, swollen fibers were present only in the deep dermal layer. Conclusion. The use of collagen bands with platelets accelerated the processes of epithelialization and collagen remodeling associated with preserved local infiltration of the wound with inflammatory cells.