Introduction. Additive technologies make it possible to create implants to replace bone defects. Determining the optimal parameters of a porous structure remains an urgent issue. The experimental research aimed to study the biocompatibility of implants made of titanium alloy with different pore diameters. Materials and methods. The study was carried out in two stages. The first stage included an in vitro study carried out on test cultures of diploid human fibroblasts to assess the cytotoxicity of the titanium alloy, from which samples were made for the second stage in vivo. The in vivo study was performed on three groups of rabbits (n=18), which received samples of the developed implants. The average age of subjects was 7±1 months, the weight being 4.675±258 g. All samples were 4 mm in diameter and 6 mm high. In group 1, the pore size was 100 μm; in group 2 – 200 μm; and in group 3 – 400 μm; the porosity was 55%, 62%, and 70%, respectively. The animals were sacrificed on day 90 after placement of the samples. Histological, morphological, and electron microscopic studies were performed to assess the osseointegrative properties of the implants. Results. In the in vitro study, we detected no toxic effects of the material on the test culture. In the in vivo study, the histological analysis did not reveal inflammation in peri-implant tissues in any of the groups. The morphological study showed a newly formed tissue in the area of the formed defect which consisted of young bone trabeculae with osteoblasts located on their surface. In group 3, we found the greatest prevalence of mature bone trabeculae. The samples with a 400-μm pore diameter revealed the largest area of filling the implant with newly formed bone tissue. Conclusion. Samples with a pore diameter of 400 μm have the highest osteointegrative properties. Keywords: osteointegration, biocompatibility, titanium implants, additive technologies, 3D-printing

The aim of the study was to develop a technology for repairing an osteomyelitic bone defect using autologous adipose tissue mesenchymal stromal cells (MSCs) bound to a collagen matrix and to test the efficacy of this technique.Materials and Methods:The study was carried out with 17 rabbits. A bone defect was created using a milling cutter applied to the proximal third of the leg. The wound (8.0×4.0 mm and a depth of 4.0 mm) involved the periosteum, cortical layer, and cancellous substance. Staphylococcus aureus strain was used as an infectious agent.After the development of chronic osteomyelitis, the animals underwent osteonecrectomy. In the study group, autologous MSCs in Collatamp EG collagen carrier were placed into the bone defect. MSCs were obtained from adipose tissue and cultured in the matrix for 5 days. In control, the defect was filled with the collagen matrix without cells.Results:On day 14 upon the initiation of chronic osteomyelitis, bacteriological examination of the discharge from the fistula showed the presence of mixed bacterial flora (Staphylococcus aureus and Escherichia coli) in all operated animals. Results of X-ray, laboratory, and histological tests confirmed the formation of a focus of chronic osteomyelitis.Two months after the treatment (collagen with or without MSCs) began, all animals of the study group showed mature bone tissue regenerated in the affected zone. In the control group, proliferation of osteoblasts on the surface of the bone trabeculae was also observed; however, mature osteoid tissue was more often detected in the study group (35.0 vs 20.0% in control). In the study group (MSCs + collagen matrix), there was a decrease in bone marrow fibrosis (50.0 vs 100.0% in control) and cartilage formation (30.0 and 66.7%, respectively). After full treatment, newly formed bone trabeculae were detected more often (100.0 vs 60.0% in control); they were more mature and filled the defect area more efficiently.Conclusion:Our results indicate that the use of a collagen matrix with autologous MSCs is a promising plastic material for repairing osteomyelitic defects following necrectomy.The MSCs were able to increase the density of the filling material in the bone cavity, significantly accelerate the formation of bone beams around the matrix, and increase the tissue volume around the implant. The presence of MSCs significantly decreased the interference of a connective tissue component with osteogenesis and chondrogenesis.

The aim of the study is to evaluate biocompatibility of a novel hybrid polyoligomer in in vitro and in vivo models. Materials and Methods. Cytotoxicity of the material was investigated using the MTT assay with human dermal fibroblasts as test cultures. To study direct interaction of the hybrid polyoligomer with cells, the fibroblasts were cultured on the polymer samples for 96 h, the cultures were assessed every 24 h using fluorescence microscopy. To study the tissue reaction in the area of contact with the donor bed and the morphological features of the implanted sample restructuring, a case-control study was performed using a rabbit model. Samples of hybrid polyoligomer were implanted into the bone defect formed in the left iliac crest in 10 rabbits. In the control group, the prepared allograft samples were transplanted into similar defects in 10 animals. The rabbits were sacrificed 4 and 8 weeks after the operation. The standard morphological methods with hematoxylin and eosin staining and immunohistochemical Ki-67 proliferation marker evaluation were used to assess the state of tissues in the defect area. Results. The results demonstrate that the hybrid polyoligomer is not cytotoxic (cytotoxicity score 0-1), cells adhere well to its surface, retain their viability and typical morphology throughout the entire observation period. No negative impact of material implantation on the health state and behavior of animals was detected. Morphological examination showed the absence of inflammatory changes, formation of thin-walled capillary vessels, and considerable proliferative activity of mesenchymal cells in the defect area, even though it was more intense than in the control group. Conclusion. No inflammation signs were detected by 8th week of the experiment. It was defined that new bone was beginning to form. The results of analysis support the conclusion that the developed hybrid materials are prospective for further research as potential bone substitute.

Objective : to develop a model of a biomedical cell product that is consistent with the «homologous drug» strategy  based on protocols for preparing the cell component and scaffold carrier for preclinical studies on a large laboratory  animal (pig). Materials and methods . Biomedical cell products and skin equivalents (SE), were formed using  plasma cryoprecipitate prepared from blood plasma of healthy donors and mesenchymal stem cells (MSCs) of  human adipose tissue. Cryoprecipitate from pig blood plasma and human adipose tissue-derived MSCs were used   to form model skin equivalents (mSE). Bright-field microscopy, phase-contrast microscopy (Leica DMI 3000B)  and fluorescence microscopy (Cytation 5 imager; BioTek, USA) were used to monitor the state of cells in the  culture and in the composition of the equivalents. Scaffolds for equivalents were tested for cytotoxicity (MTT test,  direct contact method). The cell distribution density was characterized by author’s method (Patent No. 2675376  of the Russian Federation). Results . An mSE was developed for preclinical studies on a large laboratory animal  (pig). In the mSE, components that change from halogen to xenogenic conditions during transplantation to the  animal were replaced. A comprehensive approach to preparing mSE was presented. It includes sampling of primary  pig biomaterial, extraction and characterization of adipose tissue-derived MSCs, preparation of a scaffold  carrier for the corresponding «homologous drug» strategy. Cytotoxicity of the mSE scaffold was evaluated. It  was shown that mSE provides mechanical support (similar to SE) to cells, as well as comparable development of  cellular events during cultivation. Conclusion . A model of a biomedical cell product was developed. This model  is consistent with the «homologous drug» strategy for preclinical studies on a large laboratory animal (pig). The  paper presented a comprehensive approach to developing a model equivalent based on protocols for preparation  and testing of the cellular component, the scaffold carrier and the ready-to-use model equivalent.

We present the results of studies of the interaction of cells with various materials and products from them intended for biomedical use. Widefield fluorescence microscopy enables evaluating the cytotoxicity of samples, cell viability, morphology, and proliferative activity in the process of interaction with the test samples, as well as adhesion, migration, and cell distribution both on the surface and in the structure of scaffolds and biomedical cell products. The methods of fluorescence microscopy make possible intravital studies of the interaction of cells with materials without additional processing or destruction of samples.

The results of studies representing the interaction of cells with various products provided for biomedical use are stated. It has been shown that wide-field fluorescence microscopy makes it possible to evaluate the cytotoxicity of the samples, the viability, morphology and proliferative activity of cells during the process of interaction, as well as the adhesion, migration, and distribution of cells, both on the surface of materials and in the structure of scaffolds and biomedical cell product. Methods of wide-field fluorescence microscopy make it possible to carry out intravital studies of the interaction of cells with materials without any additional processing and samples destruction.

Objective : to develop a model of a biomedical cell product that is consistent with the «homologous drug» strategy based on protocols for preparing the cell component and scaffold carrier for preclinical studies on a large laboratory animal (pig). Materials and methods . Biomedical cell products and skin equivalents (SE), were formed using plasma cryoprecipitate prepared from blood plasma of healthy donors and mesenchymal stem cells (MSCs) of human adipose tissue. Cryoprecipitate from pig blood plasma and human adipose tissue-derived MSCs were used to form model skin equivalents (mSE). Bright-field microscopy, phase-contrast microscopy (Leica DMI 3000B) and fluorescence microscopy (Cytation 5 imager; BioTek, USA) were used to monitor the state of cells in the culture and in the composition of the equivalents. Scaffolds for equivalents were tested for cytotoxicity (MTT test, direct contact method). The cell distribution density was characterized by author’s method (Patent No. 2675376 of the Russian Federation). Results . An mSE was developed for preclinical studies on a large laboratory animal (pig). In the mSE, components that change from halogen to xenogenic conditions during transplantation to the animal were replaced. A comprehensive approach to preparing mSE was presented. It includes sampling of primary pig biomaterial, extraction and characterization of adipose tissue-derived MSCs, preparation of a scaffold carrier for the corresponding «homologous drug» strategy. Cytotoxicity of the mSE scaffold was evaluated. It was shown that mSE provides mechanical support (similar to SE) to cells, as well as comparable development of cellular events during cultivation. Conclusion . A model of a biomedical cell product was developed. This model is consistent with the «homologous drug» strategy for preclinical studies on a large laboratory animal (pig). The paper presented a comprehensive approach to developing a model equivalent based on protocols for preparation and testing of the cellular component, the scaffold carrier and the ready-to-use model equivalent.

Polymer monoliths containing open interconnected pores were synthesized by a visible-light induced polymerization of oligocarbonatedimethacrylate (OCM-2) in the presence of dialkylphthalates (C6H4[C(O)OR](2), R = C2H5, n-C4H9, n-C8H17 and n-C9H19) as porogenic agent. A closeness of the refractive indices of the forming polymer and dialkylphthalates provides for the formation of transparent porous monoliths, thus making manufacture of thick porous polymeric materials using visible light possible. The porous polymers derived from OCM-2 can sorb water and benzene. The filling of pores by water increases with an increase of a number of carbon atoms in the ester groups from 0.65 for diethyl phthalate to 1.0 for dinonyl phthalate. Experimentally defined value of Hildebrand solubility parameter delta for poly-OCM-2 is 24.9 MPa1/2. A closeness of the solubility parameters of the polymer (delta(p)) and porogenic agent (delta(s)) guarantees the porous architecture of the polymer formed. The porous polymeric materials have been studied in vitro using a culture of human dermal fibroblasts (HDF) of four passages. The MTT test (MTT is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) has shown that the materials are not cytotoxic. During long-term cultivation of the cells on the material surface a pronounced cell adhesion as well as cell viability and proliferative activity have been detected.

The aim of the study was the development of methods to assess the quality of biomedical cell products (BMCPs) intended to replace skin defects. Materials and Methods. The proposed equivalent of the skin BMCP-1 (developed at the N.K. Koltsov Institute of Developmental Biology, Russian Academy of Sciences) and the BMCP-2 equivalent of the skin (developed at the Privolzhsky Research Medical University) were studied. Mesenchymal stem cells (MSCs) from human adipose tissue served as the cellular components of both BMCPs. MSCs in suspensions and in BMCPs were tested for cell counts and cell viability. The BMCPs were studied in their entirety without destruction using fluorescence microscopy with vital dyes for staining the cytoplasm and Hoechst 3334 (BD Pharmingen, USA) — for nuclei (imager Cytation 5; BioTek, USA). The MSC function was evaluated by their ability to produce VEGF-A. The MSC phenotype was determined by cytometry. Results. Using the above methods, we found that MSCs in BMCP retained their original morphology and viability. On the surface of BMCP-1, cells are organized in colonies, whereas in the structure of BMCP-2, they are scattered throughout the matrix. The number of cells in BMCP-1 depends on the transportation conditions; and in the structure of BMCP-2 — on the timing of cultivation. The secretory activity of MSCs is maintained throughout the entire observation period. While within the BMPC structures, the MSCs had their CD90 expression decreased; it was then restored after the cells were isolated from the products and cultivated on the plastic surface. Conclusion. The proposed method is feasible for the BMCP quality assessment; it incorporates the requirements for production and transportation based on the characteristics of the cellular and non-cellular components. Given the optical non-transparency and complex physical-chemical structure of the product, it is advisable to select the quality control methods that ensure minimal manipulation and enzymatic damage.

e-mail: daleynik@yandex.ru Burn injury with a lesion of a large area of the body surface remains a serious problem for surgeons. Every year In Russia about 400 thousands of people receive burns of different severity level, among which about 30 % of people need hospitalization. In recent decades, methods of regenerative medicine have been actively used to treat burn injuries. One of the promising areas to treat burns are technologies based on the use of uncultivated freshly harvested autologous skin cells. The experience of treating of 14 patients with extensive burns of 20–80 % is presented. To restore the lost of skin cover a suspension of freshly isolated autologous skin cells in combination with a 1: 6 mesh graft and fibrin glue was used. The suspension of cells was obtained by gentle cold trypsinization. It is shown that keratinocytes and fibroblasts are included in the obtained cell suspension, cells’ viability is more than 60 % and cells’ yield from 1 cm 2 is about 6 million. The completion of epithelialization in the cells of the autografts within 6-14 days after cells’ transplantation is recorded. Following observation for the patients within 12 months after discharge form the hospital showed satisfactory cosmetic and

The purpose of the study is to assess the possibility of applying the integrated module as the basis of a cell-tissue equivalent for treatment of wounds of skin and soft tissues. In the frame of the set task the following problems were being solved: research of the spatial structure and architectonics of the surface of the developed base collagen-containing materials and their biocompatibility with cell cultures.Materials and methods. The study of a material which is a two-layer complex film, consisting of collagen and polysaccharide components was carried out. The collagen was separated from the dermis and was then impregnated with particulate demineralized bone matrix (DCM) according to the original methodology. For the purposes of the study the dehydrated material was created in the form of a film. Electron microscopic examination of surfaces was performed on scanning electron microscope JEOL JSM-IT300LV in high vacuum and at low values of probe current (< 0,1 nA). Studies to assess the viability of the cells cultivated on films of collagen material (tested for cytotoxicity and the adhesive capacity) were performed in vitro using strains of diploid human fibroblasts 4-6 passage. The culture condition was visually assessed using an inverted Leica microscope DM IL (Carl Zeiss, Austria), equipped with a computerizes program of control of culture growth (Leica IM 1000).Results. The data obtained in the study of the surface structure of the developed complex module showed that it seems to be promising as a basic component of the cellular-tissue system with its large number of structural formations for fixation of the cells and a well-organized barrier layer capable of vapor - permeability. Experiments in vitro confirmed the absence of toxicity of the material being studied in relation to the culture of dermal human fibroblasts, suggesting the possibility of creation on its basis of cell-tissue complex and further experimental studies in vivo.Conclusion. Thus it was experimentally confirmed that the physical characteristics of the developed integrated module satisfy the requirements for the materials for cultivation of cells. The absence of cytotoxicity on the model of a culture of dermal human fibroblasts allows to make a conclusion about a possibility of its use as the basis of cell-tissue equivalent. Preliminary results indicate the advisability of further experiments in vivo aimed at improving complex collagen-containing materials, the development of different ways of their application and clinical evaluation of the effectiveness in the treatment of wounds of various genesis.

A new procedure for obtaining nanosized hydroxyapatite (Ca 5(PO 4) 3OH) powder is presented. The obtained compound has been tested on a human fibroblast cell culture to clarify its biological compatibility and activity. The synthesized hydroxyapatite is shown to be non-toxic to human dermal fibroblast cultures in vitro.

Цель. Оценить выживаемость больных ХСН, страдающих СД 2; выявить параметры диабета, влияющие на течение и прогноз пациентов с недостаточностью кровообращения. Материалы и методы. Проведено исследование когорты дожития, которую составили 72 больных ХСН, страдающих СД 2. Судьба больных прослежена в течение 12 мес. У всех больных был собран анамнез и проведено физикальное обследование. Производилась оценка ФК ХСН. Выполнено Эхо-КГ. Исследовали уровень предсердного натрийуретического пептида (ПНУП). Результаты. Всего за 12 мес. наблюдения скончались 17 больных. Достоверно с выживаемостью связаны показатели тяжести ХСН, отражающие переносимость нагрузки (функциональный класс), клиническую характеристику (ШОКС) и нейрогуморальную активацию (ПНУП). Уровень ПНУП, более чем в 4 раза превышающий норму (т.е. более 8000 фмоль/мл), явился мощным предиктором неблагоприятного исхода в течение одного года. В группе выживших больные со ?стажем? СД более 10 лет составили 43% против 76% в группе скончавшихся. В группе больных с уровнем НвА1с менее 6,5% в половине случаев отмечались признаки хронической почечной недостаточности. При ХПН снижается активность почечной инсулиназы. достоверно на выживаемость оказывали влияние тяжесть ХСН и наличие уремической стадии диабетической нефропатии. На каждый балл увеличения ШОКС риск смерти в течение года возрастает на 25%. Наличие ХПН увеличивает риск смерти в 3 раза. Выводы. Почти 1/4 часть больных ХСН, страдающих СД 2, погибает в течение года. Существенное влияние на выживаемость оказывает исходная тяжесть ХСН. Наибольшее значение для прогноза среди характеристик СД 2 имеет диабетическая нефропатия. Ухудшение выживаемости отмечено уже на стадии микроальбуминурии. При наличии уремии риск смерти возрастает в 3 раза. Для быстрой оценки риска смерти в течение года можно ориентироваться на ШОКС и на наличие признаков ХПН. Для улучшения прогноза больных ХСН, страдающих СД 2, необходимо особое внимание уделять профилактике, ранней диагностике и лечению диабетического поражения почек.

Morphological analysis and morphometry using immunoperoxidase methods showed that microwave (MW) radiation with a white noise type spectrum and radiation power density close to that emitted by the object stimulated proliferation of human fibroblasts in vitro . This effect is realized via stimulation of DNA synthesis. MV irradiation with the same parameters increased adhesive capacity of human blood neutrophils.