Method for preserving the viability of a chicken embryo with a shell defect in experiment

The egg has always been and remains an ideal object for conducting various scientific research. An egg is an isolated egg cell outside the mother’s body. Therefore, it is an ideal object for studying embryogenesis and performing various manipulations during embryogenesis and before the birth of a viable organism. The existing methods allow conducting experimental manipulations with the embryo in  situ, inside the egg shells without damaging them. However, the achievement of ideal parameters for closing the defect of the fertilized egg shell in the experiment is the key to  the successful completion of the experiment. Periods of embryogenesis, especially at the last stage, when osteogenesis occurs, require the presence of a sufficient amount of calcium ions in the metabolism of the developing chicken, which are vital for the formation of a full-fledged chicken.The aim: to develop an optimal method for closing the defect and fixing the egg shell after manipulation or during the experiment.Materials and methods. The experiment was carried out on fertile eggs of the breed of chickens – meat breed of broilers Ross-308 (ROSS-308), JSC Poultry Farm “NovoBaryshevskaya” (Koltsovo, Novosibirsk Region, Russian Federation). In the experiment, 120 fertilized eggs were used. Eggs weighing 60–70 g were incubated at a temperature of 37.5–38.0 °C and 50–55 % humidity. Comparative anatomical and physiological parameters were evaluated on the 7th, 14th, 20th day of incubation and on the 1st day of the postnatal period. In the experimental group, the shell defect was covered with a fragment of the egg shell of the donor. Incubation was carried out in an incubator – a household incubator “Nesushka” (Novosibirsk, Russian Federation).Results. The proposed method of fixing and closing the defect of the fertilized egg shell excludes the use of foreign materials that have an adverse effect on the development of the embryo. There were no anatomical and physiological deviations in the chicks of the study group when comparing the indicators with the parameters in the comparison group and the Hamburger – Hamilton classification

IMPROVEMENT OF SPENT NUCLEAR FUEL COOLING SAFETY IN COOLING PONDS USING THERMOSIPHONS

В работе рассмотрен способ пассивного отвода остаточных тепловыделений отработавшего ядерного топлива (ОЯТ) в бассейне выдержки с помощью термосифонов для повышения безопасности в случае потери электроснабжения.The work considers a way of passive removal of residual heat from spent nuclear fuel in a cooling pond with the use thermosyphons in order to improve safety in case of power supply failure

Possibility of using the infrapatellar (Hoffa’s) fat pad as a source of autologous stem cells

Stem cells are the basis for the creation of tissue-engineered structures in regenerative medicine. The most well-studied sources of stem cells are the embryo and bone marrow. The use of embryonic cells is associated with ethical problems, and the collection of bone marrow is accompanied by invasive procedures. Using adipose tissue as a source of stem cells avoids these problems. But the collection of adipose tissue requires additional interventions, which does not exclude the occurrence of cosmetic defects. Aim of the study was to investigate the possibility of using mesenchymal stem cells (MSCs) isolated from the infrapatellar (Hoffa’s) fat pad.Material and methods. As a source of MSCs, tissue samples of Hoffa’s fat pad removed during the operation were used (8 cases), as a control - MSCs isolated from human adipose tissue (6 cases). MSCs were isolated using an enzymatic method. At the 3rd passage, phenotyping with specific antibodies against CD34, CD45, CD73, CD90, CD105 was performed by flow cytometry. Differentiation in the chondro- and osteogenic direction was carried out at the 3rd passage with the appropriate differentiation media. Chondrogenic differentiation was confirmed by staining with alcian blue, osteogenic - staining according to von Kossa.Results and discussion. Statistically significant decrease in CD105 expression, increase in CD73, CD34 expression and lack of adequate differentiation under standard conditions of differentiation media by MSCs isolated from the Hoffa’s fat pad compared to control was found. The data obtained indicate a discrepancy between the cells isolated from the Hoffa’s fat pad and the requirements for MSCs.Conclusion. The infrapatellar (Hoffa’s) fat pad_cannot be used as a source of standardized MSCs

Properties Of The Hard X-ray Radiation From The Black Hole Candidates: Cygnus X-1 And 1E1740.7-2942

The entire dataset of the GRANAT/SIGMA observations of Cyg X-1 and 1E1740.7-2942 in 1990-1994 was analyzed in order to search for correlations between primary observational characteristics of the hard X-ray (40-200 keV) emission - hard X-ray luminosity, hardness of the spectrum (quantified in terms of the best-fit thermal bremsstrahlung temperature kT) and the RMS of short-term flux variations. Although no strict point-to-point correlations were detected certain general tendencies are evident. It was found that for Cyg X-1 the spectral hardness is in general positively correlated with relative amplitude of short-term variability. The correlation of similar kind was found for X-ray transient GRO J0422+32 (X-ray Nova Persei 1992). For both sources an approximate correlation between kT and L_X was found. At low hard X-ray luminosity - below 10E37 erg/sec - kT increases with L_X. At higher luminosity the spectral hardness depends weaker or does not depend at all on the hard X-ray luminosity. The low luminosity end of these approximate correlations (low kT and low RMS) corresponds to extended episodes of very low hard X-ray flux occurred during SIGMA observations.Comment: 6 pages, 7 figures, uses mn.sty, epsf.sty, psfig.tex; Accepted for publication in MNRAS; Also available at http://hea.iki.rssi.ru/~sik/PAPERS/mnras97.ps.gz (gzipped PostScript

Using Relational Verification for Program Slicing

Program slicing is the process of removing statements from a program such that defined aspects of its behavior are retained. For producing precise slices, i.e., slices that are minimal in size, the program\u27s semantics must be considered. Existing approaches that go beyond a syntactical analysis and do take the semantics into account are not fully automatic and require auxiliary specifications from the user. In this paper, we adapt relational verification to check whether a slice candidate obtained by removing some instructions from a program is indeed a valid slice. Based on this, we propose a framework for precise and automatic program slicing. As part of this framework, we present three strategies for the generation of slice candidates, and we show how dynamic slicing approaches - that interweave generating and checking slice candidates - can be used for this purpose. The framework can easily be extended with other strategies for generating slice candidates. We discuss the strengths and weaknesses of slicing approaches that use our framework

ЗАСЕЛЕНИЕ ДЕМИНЕРАЛИЗОВАННОГО КОСТНОГО МАТРИКСА КЛЕТКАМИ ХОНДРОГЕННОГО РЯДА

Aim To determine optimal approaches of demineralized bone tissue processing after preservation to ensure efficient seeding of chondrocytes. Methods Demineralized bone matrix specimens sized 1 x 1 x 1 cm3 were used in the experiment. A purification method ensuring the removal of cytotoxic substances from the matrices has been developed. It consists of a multi-stage soaking of the specimens in H2O, 0.1H NaOH, 1N NaOH, H2O and DPBS until a neutral pH is reached. After chemical purification (a 3-stage process), all the specimens were subjected to sonication for 1 minute at 5W to improve cell adhesion. The water was changed after each exposure. Then, the water was replaced to DPBS and the specimens were sonicated for 1 minute at 5W. After it, the sample was placed in a neutral medium (pH 7.0). The matrices undergoing sonicated procession were seeded with cells. Hyaline cartilage of minipigs was used as a source of the cells. Chondrocytes were isolated using collagenase II digestion and cultured for 20 days in the culture flasks. Passage 1 chondrocytes were seeded on the matrices. DBM were pretreated with a 1% gelatin solution to improve the efficiency of cell seeding. The microtitration viability test estimating the impact of the extract obtained during sonation cycles on cell viability was performed to determine whether these matrices may be seeded with chondrocytes. The test was performed on the lag- and log-phase cells. The effect of the extract on the cells lasted around 3 days. Results Extract-treated chondrocytes during the lag-phase showed a direct dose-dependent cytotoxic effect, compared to extract-treated chondrocytes during the log-phase. Low efficiency of DBM was associated with both, the stringent requirements for the manufacturing process of DBM and the subsequent matrices processing, including the cell growth phases. The increased cell migration depth into the matrices resulted in the disturbances of the microcirculation, leading to the insufficient cell feeding and slowed down metabolic processes. Conclusion The efficiency of DBM cell seeding depends on the matrix processing, its cytotoxic effect and architectonics. The problem of slowing down the metabolism of cells in DMB may be solved by the application of the combined purification technique, i.e. chemical and ultrasonic purification methods. The obtained results prove the necessity of using mechanical and electrical stimuli for the normal functioning of bone and cartilage tissue cells within the matrix.Цель Поиск эффективных способов обработки деминерализованной костной ткани после консервации для эффективного заселения хондроцитами. Материалы и методы В качестве материала исследования использовали деминерализованный костный матрикс размером 1 х 1 х 1 см³. Для удаления цитотоксических веществ из матриц разработан способ очистки, заключающийся в поэтапном замачивании образца в Н2О, растворе 0,1 Н NaOH, растворе 1 н NaOH, Н2О и DPBS до нейтрального pH. Для улучшения клеточной адгезии на матрицах, на последние перед заселением воздействовали ультразвуком. На образец, прошедший химическую очистку 3 раза, воздействовали ультразвуком в течение 1 минуты и W = 5. После каждого воздействия, воду в емкости меняли. После этого воду в емкости сменили на DPBS и обрабатывали ультразвуком в течение 1 минуты и W = 5. После окончания процедур образец находился в нейтральной среде (pH 7,0). Обработанные таким способом матрицы заселяли клетками. В качестве источника клеток для заселения, была выбрана ткань гиалинового хряща мини-поросенка. Хондроциты выделяли стандартным способом с применением коллагеназы II типа и культивировали в течение 20 суток в культуральных флаконах. Матрицы заселяли хондроцитами 1 пассажа. Для повышения эффективности заселения костного матрикса клетками был апробирован способ предварительной обработки деминерализованной костной ткани 1% раствором желатина. Для определения пригодности матрикса к заселению его хондроцитами использовали микротитрационный тест влияния экстракта, получаемого в ходе ультразвуковой обработки матрицы, на жизнеспособность клеток. Тест проводили на лаг- и лог-фазах роста клеток. Воздействие экстракта на клетки длилось 3 суток. Результаты Показано, что обработка хондроцитов экстрактом на этапе лаг-фазы роста культуры оказывает прямой дозозависимый цитотоксический эффект, в отличие от эффекта обработки хондроцитов в фазе логарифмического роста культуры. Показано, что низкая эффективность заселения деминерализованного костного матрикса связана не только с жесткими условиями изготовления ДКМ, но и от условий последующей подготовки матрицы, фазы роста заселяемой клеточной культуры. С увеличением глубины миграции клеток вглубь матрицы нарушается микроциркуляция, что ведет к недостаточному обеспечению клеток в тканеинженерной конструкции питанием и замедлению метаболических процессов. Заключение Эффективность равномерного заселения деминерализованного костного матрикса клетками связана не только с условиями обработки матрикса, выраженным цитотоксическим эффектом, но и с его архитектоникой. Проблема замедления метаболизма клеток в тканеинженерной конструкции решается путем комбинированного метода очистки ДКМ химическим и ультразвуковым способом. Полученные результаты свидетельствуют о необходимости применении механических и электрических стимулов для нормального функционирования клеток костной и хрящевой ткани внутри матрицы

ОСОБЕННОСТИ РЕГЕНЕРАЦИИ КОСТНОЙ ТКАНИ ТЕЛ ПОЗВОНКОВ НА ОСНОВЕ ОСТЕОТРАНСПЛАНТАТА В ЭКСПЕРИМЕНТЕ

Aim. To assess osteogenic potentialities of a three-di-   components, enzymes and vessels with endothelial lining. mensional osteograft in a mini pig model with artificial   Structural composition of the osteograft is an analogue of vertebral body defect. The osteograft consists of osteo-               embryonic bone tissue, which was the basis for examining the regenerative potentialities of the osteograft in the ex-periment.Methods. The osteograft was implanted in the defect of the lumbar vertebral body of minipig (6 months old). The animals were withdrawn from the experiment in the period from 14 days to 6 months. In the control series, autobone was implanted in a similar defect. The preparations were examined by morphological methods, and bone tissue density was assessed by MSCT.Results. Regeneration and integration of the bone tissue of the vertebral body with the defect replaced by the osteograft was by primary angiogenic osteogene sis within one month due to the structural components of the osteograft. When the defect is replaced by autograft, the regeneration and integration of the bone tissue of the vertebral body occur within six months due to the structural components of the recipient.Conclusion. Formation of the common blood flow of the transplant and recipient vessels is both a factor of integration of the transplant into the homeostatic system of the recipient, and a pathogenic mechanism for optimizing the regeneration of bone tissue defect on the basis of the graft.Цель. На экспериментальной модели артифициального дефекта тела позвонка мини-поросенка исследованы остеогенные потенции трехмерного остеотрансплантата. Остеотрансплантат состоит из клеток остеогенного ряда, матрикса, содержащего костные белки, минеральные компоненты, ферменты и сосуды с эндотелиальной выстилкой. Структурная композиция остетрансплантата является аналогом эмбриональной костной ткани, что явилось основанием для исследования регенераторных потенций остеотрансплантата в эксперименте.Материалы и методы. В дефект тела поясничного позвонка мини-поросенка (6 месяцев) имплантировали остеотрансплантат. Животных выводили из эксперимента в сроки от 14 дней до 6 месяцев. В контрольной серии в подобный дефект имплантировали аутокость. Препараты исследова ескими методами, плотность костной ткани оценивали методом МСКТ.Результаты. Регенерация и интеграция костной ткани тела позвонка при замещении остеотрансплантатом происходит первичным ангиогенным остеогенезом за счет структурных компонентов остеотрансплантата в течение одного месяца. Регенерация и интеграция  сплантатом происходит за счет структурных компонентов реципиента в течение шести месяцев.Выводы. Формирование общего кровотока трансплантата и сосудов реципиента является фактором интеграции трансплантантата в гомеостатическую систему реципиента и патогенетическим механизмом оптимизации регенерации дефекта костной ткани на основе трансплантата

СВОЙСТВА ДЕМИНЕРАЛИЗОВАННОГО КОСТНОГО МАТРИКСА ДЛЯ БИОИНЖЕНЕРИИ ТКАНЕЙ

The purpose. Determination of tissues of physico-mechanical properties of demineralized bone matrix of spongy and compact human bone important for bioengineering.Material and Methods.The methods for studying micromorphological, piezoelectric and transport properties, adapted for measuring the materials of potential scaffolds.Results. The results of studying the physico-mechanical properties of the demineralized bone matrix of spongy and compact human bones are presented. It is shown that the demineralized spongy bone possesses the best characteristics of the pore system for the colonization of matrix cells. The tensile strength and modulus of elasticity of samples from the demineralized heads of the femurs extracted during the initial hip arthroplasty vary widely. The modulus of elasticity varied from 50 to 250 MPa, and the ultimate strength was from 1.1 to 5.5 MPa.Conclusion. Methods for measuring micromorphological, piezoelectric and transport properties for materials of potential matrices were developed and / or adapted. It is shown that in the samples of materials from the human bone, these characteristics, as a rule, vary considerably. Proceeding from this, it becomes obvious that the development of protocols of measurement methods of the above listed properties is an important work for the creation of technology of bioengineering of tissue implants for reconstructive surgery. Цель. Определение значимых для биоинженерии тканей физико-механических свойств деминерализованного костного матрикса губчатой и компактной кости человека.Материалы и методы. Перечислены методы исследования микроморфологических, пьезоэлектрических и транспортных свойств, адаптированные для измерения у материалов потенциальных матриц.Результаты. Приведены результаты исследования физико-механических свойств деминерализованного костного матрикса губчатой и компактной кости человека. Показано, что деминерализованная губчатая кость обладает наилучшими характеристиками поровой системы для заселения матриксов клетками. Предел прочности и модуль упругости образцов из деминерализованных головок бедренных костей, извлеченных в ходе первичного эндопротезирования тазобедренного сустава, изменяются в широких пределах. Модуль упругости изменялся от 50 до 250 МПа, а предел прочности – от 1,1 до 5,5 МПа.Заключение. Были отработаны и/или адаптированы методы измерений микроморфологических, пьезоэлектрических и транспортных свойств у материалов потенциальных матриц. Показано, что у образцов материалов из кости человека данные характеристики, как правило, значительно варьируют. Исходя из этого, становится очевидным, что отработка протоколов методов измерения вышеперечисленных свойств является важной работой для создания технологии биоинженерии тканевых имплантатов для восстановительной хирургии.

Nanoencapsulated capsaicin changes migration behavior and morphology of madin darby canine kidney cell monolayers

We have developed a drug delivery nanosystem based on chitosan and capsaicin. Both substances have a wide range of biological activities. We investigated the nanosystem’s influence on migration and morphology of Madin Darby canine kidney (MDCK-C7) epithelial cells in comparison to the capsaicin-free nanoformulation, free capsaicin, and control cells. For minimally-invasive quantification of cell migration, we applied label-free digital holographic microscopy (DHM) and single-cell tracking. Moreover, quantitative DHM phase images were used as novel stain-free assay to quantify the temporal course of global cellular morphology changes in confluent cell layers. Cytoskeleton alterations and tight junction protein redistributions were complementary analyzed by fluorescence microscopy. Calcium influx measurements were conducted to characterize the influence of the nanoformulations and capsaicin on ion channel activities. We found that both, capsaicin-loaded and unloaded chitosan nanocapsules, and also free capsaicin, have a significant impact on directed cell migration and cellular motility. Increase of velocity and directionality of cell migration correlates with changes in the cell layer surface roughness, tight junction integrity and cytoskeleton alterations. Calcium influx into cells occurred only after nanoformulation treatment but not upon addition of free capsaicin. Our results pave the way for further studies on the biological significance of these findings and potential biomedical applications, e.g. as drug and gene carriers