Совместное использование разработанных коллагенсодержащих комплексов и культуры клеток для создания новых тканевых эквивалентов

The purpose of the study is to assess the possibility of applying the integrated module as the basis of a celltissue 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 nА). 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.Цель исследования – оценка возможности применения созданного комплексного модуля в качестве основы клеточно-тканевого эквивалента для лечения ран кожи и мягких тканей. В рамках поставленной цели решались задачи по изучению пространственной структуры и архитектоники поверхности разработанных базовых коллагенсодержащих материалов и их биосовместимости с клеточными культурами.Материал и методы. Проведено исследование материала, который представляет собой двухслойный пленочный комплекс, состоящий из коллагенового и полисахаридного компонентов. Коллаген выделялся из дермы и был импрегнирован мелкодисперсным деминерализованным костным матриксом (ДКМ) по оригинальной методике. Для исследования дегидратированный материал был выполнен в виде пленки. Электронно-микроскопическое исследование поверхности проведено на сканирующем электронном микроскопе JEOL JSM-IT300LV (Япония) в режиме высокого вакуума и при низких значениях тока зонда (< 0,1 нА). Исследования по оценке жизнеспособности клеток в условиях культивирования на пленках из коллагенсодержащего материала (тестирование на цитотоксичность и адгезивную способность) проводили in vitro с использованием штаммов диплоидных фибробластов человека 4–6-го пассажа. Состояние культуры оценивали визуально с помощью инвертированного микроскопа Leica DM (Carl Zeiss, Австрия), оснащенного компьютерной программой контроля роста культуры (Leica IM 1000).Результаты. Данные, полученные при изучении структуры поверхности разработанного комплексного модуля, показали, что он представляется перспективным в качестве базового компонента клеточно-тканевой системы: наличие большого количества структурных образований для фиксации клеток и хорошо организованного барьерного слоя, способного к паро- и водопроницаемости. Таким образом, экспериментально подтверждено соответствие физических характеристик разработанного базового комплекса требованиям, предъявляемым к материалам для культивирования клеток. Показано отсутствие цитотоксичности на модели культуры дермальных фибробластов человека, что позволяет сделать вывод о возможности его использования в качестве основы клеточно-тканевого эквивалента. Полученные предварительные результаты указывают на целесообразность дальнейшего проведения экспериментов in vivo, направленных на совершенствование комплексных коллагенсодержащих материалов, разработку различных способов их применения и клиническую оценку эффективности при лечении ран различного генеза

Joint use of developed collagen-containing complexes and cell cultures in creating new tissue equivalents

The purpose of the study is to assess the possibility of applying the integrated module as the basis of a celltissue 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 nА). 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

Prediktory vyzhivaemosti bol'nykh khronicheskoy serdechnoy nedostatochnost'yu, stradayushchikh sakharnym diabetom 2 tipa

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

In vitro dendritic cell maturation isolated from healthy people and patients with Staphylococcus aureuscaused chronic osteomyelitis

Here we present the data comparing maturation of peripheral blood mononuclear cell-derived dendritic cells (DCs) isolated from healthy volunteers and Staphylococcus aureus-positive patients with chronic osteomyelitis. Dendritic cells were cultured in a standard maturation cell medium (RPMI-1640,  supplemented with antibiotics, L-glutamine, 15% calf embryonic serum) added with interleukin-4 and granulocyte-macrophage colony-stimulating factor, followed by adding a stimulating factor cocktail containing interleukin-1β, tumor necrosis factor-α, interleukin-6, and prostaglandin E2. Dendritic cell maturation was analyzed by estimating visual characteristics under Zeiss ODSERVER.Z1 inverted microscope using Axiovision Rel.4.8 imaging software as well as light and phase-contrast microscopy at magnification of ×40, ×100, ×200, ×400, ×630. Dendritic cell immunophenotyping was carried out by using a panel of anti-human monoclonal antibodies: anti-CD80 FITC-conjugated, anti-CD86 (B7–2) PE-conjugated, anti-HLA-DR PC7-conjugated (all from Beckman Coulter, USA), anti-CD14 PerCP-Cy5.5-conjugated, anti-CD83 APC-conjugated, anti-CD40 PE-Cy7-conjugated (Becton Dickinson, USA) as well as isotype-matched control antibodies on the FACS Canto II f low cytometer (Becton Dickinson, USA). It was shown that while maturation dendritic cells derived both from patients or volunteers increased in size and underwent dendrite formation. Moreover, expression of CD86, CD83, CD80, and CD40 markers on dendritic cells derived from patients vs. volunteers was lowered. However, DC stimulation resulted in significantly increased percentage of DCs positive for CD83 DCs co-stimulation molecules CD86, CD80, CD40 chronic osteomyelitis. However, such differences found in immature DCs in both groups disappeared upon maturation, so that expression of the key markers on day 10 was maintained at close level. In particular, expression of CD83 differentiation marker and the CD80 co-stimulation molecule on DCs from patients vs. volunteers was increased stronger. Thus, a maturation potential in DCs isolated from patients with Staphylococcus aureus-caused chronic osteomyelitis was not impaired in vitro. The data obtained open up an opportunity to use dendritic cells as a natural adjuvant-substituting component for development of individualized vaccines in treatment and prevention of recurrent chronic osteomyelitis

Biological Characteristics of Polyurethane-Based Bone-Replacement Materials

A study is presented on four polymers of the polyurethane family, obtained using a two-stage process. The first composition is the basic polymer; the others differ from it by the presence of a variety of fillers, introduced to provide radiopacity. The fillers used were 15% bismuth oxide (Composition 2), 15% tantalum pentoxide (Composition 3), or 15% zirconium oxide (Composition 4). Using a test culture of human fibroblasts enabled the level of cytotoxicity of the compositions to be determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, along with variations in the characteristics of the cells resulting from their culture directly on the specimens. The condition of cells on the surfaces of the specimens was assessed using fluorescence microscopy. It was shown that introducing 15% bismuth, tantalum, or zinc compounds as fillers produced a range of effects on the biological characteristics of the compositions. With the different fillers, the levels of toxicity differed and the cells’ proliferative activity or adhesion was affected. However, in general, all the studied compositions may be considered cytocompatible in respect of their biological characteristics and are promising for further development as bases for bone-substituting materials. The results obtained also open up prospects for further investigations of polyurethane compounds