Tensiometric estimation of material properties of tissue spheroids

Tissue spheroids have been proposed to use as building blocks in biofabrication and as bioinks in 3D bioprinting technologies. Tissue fusion is an ubiqious phenomenon during embryonic development. Biomimetic tissue spheroid fusion is a fundamental constructional principle of emerging organ printing technology because closely placed tissue spheroids could fuse into tissue and organ-like constructs in fusion permissive bioprintable hydrogel. From physical point of view tissue spheroids could be considered as a visco-elastic-plastic soft matter or complex fluid. We hypothesize that quantitative estimation of material properties of tissue spheroids using tensiometry could predict their tissue spreading and tissue fusion behavior as well as provide a powerful insight about possible speed of post-printed tissue and organ-like constructs compaction and maturation. Tissue spheroids from human fibroblasts, ovine and human chondrocytes and immortalised human keratinocytes have been biofabricated using non-adhesive cell culture plates (Corning, USA). For estimation of material properties of tissue spheroids commercial tensiometer Microsquisher have been emploied (CellScale, Toronto, Canada). Modulus of elasticity of tissue spheroids have been calculated based on peformed tissue compression tests. In order to identify structural determinants of material properties of tissue spheroids standard perturbants of cytoskeleton such as Cytochalasin D (Sigma, USA) for disruption of microfilaments and Nocodazole (Sigma, USA) for disruption of microtubules have been used. Viability of tissue spheroids have been also estimated and their morphology have been analysed using light microscopy, histochemistry, immunohistochemistry, semithin sections stained wih toluidine blue and transmission and scanning electron microscopy. Kinetics of tissue spheroids spreading on electrospun polyurethane matrices have been analysed. Kinetics of two closely placed tissue spheroids fusion have been analysed in hanging drop. Additionally toxic effect of water solution of paramagnetic gadolinium salt (Omniscan®, GE Health Care, USA) on material properties of tissue spheroids have been investigated. It have been demonstrated that material properties of tissue spheroids biofabricated from different cell types have different modulus of elasticity. Even tissue spheroids biofabricated the same cell types but from different species have different material properties. Incubation with Cytochlasin D dramatically reduces estimated material properties of tissue spheroids. Incubation with Nocodazole does not significantly change material properties of tissue spheroids. Material properties of tissue spheroids from chondrocytes (chondrospheres) correlates very well with increasing deposition and accumulation of extracellular matrix (confirmed by expression of collagen type II and glycosoaminoglycans). The incubation with toxic concentration of gadolinium solution dramatically reduces material properties of chondrospheres. There is no any significant correlation between material properties of tissue spheriods and their spreading kinetics. However, there is a certain correction between material properties of tissue spheroids and their tissue fusion kinetics. Our data demonstrate that beside already well established role of cell adhesion receptors such as cadherin and integrins in the realisation of cell cohesion inside tissue spheroids the structural determinants of material properties of tissue spheroids also include components of cytoskeleton such as actin micofilaments and accumulated extracellular matrix. It is possible to predict post-printing tissue fusion behaviour of tissue spheroids based on preliminary estimation of their material properties. Finally, it have been also shown that material properties of tissue spheroids correlate with their viability. Thus, tensiometry is a valuable method for systematic characterization of material properties of tissue spheroids and for prediction of tissue spheroids post-printed tissue fusion behaviour

Сравнительное исследование процесса хондрогенной дифференцировки мезенхимальных стромальных клеток, выделенных из разных источников

Introduction. As an alternative to autochondral transplantation, variants of chondrocyte replacement with mesenchymal stromal cells (MSCs) were considered, since these cells are present in all organs and tissues of human body and possess multilinear potential for differentiation. A number of studies demonstrate that the ability for chondrogenic differentiation of MSCs from different tissues varies, however, those studies are few and controversial. In accordance with the ethical principles and the technical ease of obtaining, adipose tissue, Wharton’s jelly (stroma) and dental pulp are the most attractive sources of MSCs for tissue engineering.Aim: to compare the chondrogenic potential of MSCs allocated from adipose tissue, Wharton’s jelly of the umbilical cord, and human deciduous teeth pulp cultured in the composition of microspheres (pellets).Materials and methods. The phenotype of primary cultures of MSCs was studied via flow microscopy. Chondrogenic differentiation was performed with 3D-culture in microspheres in the presence of TGFβ1 for two weeks under standard conditions. Human chondroblasts were used as a positive control. Cell viability was determined by fluorescent staining. Morphological study was performed using histological and immunohistochemical staining methods.Results. Cultures of MSCs from all sources had similar phenotypes CD29+, CD34–, CD44+, CD49b+, CD45–, CD73+, CD90+, HLADR. In the mass of living cells in microspheres only singular dead cells were visualized. A significant production of extracellular matrix (ECM) was observed in the chondroblast and adipose tissue microspheres, whereas in the microspheres of dental pulp and umbilical cord stroma derived MSCs, no significant ECM was detected. Among the MSCs, the highest production of collagen and glycosaminoglycans (GAG) in the ECM was observed in the microspheres of adipose tissue-derived MSCs, and the lowest production – in the microspheres of dental pulp-derived MSCs. Conclusion. A histological analysis of all microspheres after 14 days of culturing in the chondrogenic medium revealed the signs of differentiation in the chondrogenic direction, a progressive increase of the ECM produced by cells, and the presence of total collagen and GAG in it. Of all the MSCs studied, the greatest chondrogenic potential in vitro (the intensity of development of ECM components) is possessed by the adipose tissue-derived MSCs.Введение. В качестве альтернативы аутохондротрансплантации рассматриваются варианты замены хондроцитов на мезенхимальные стромальные клетки (МСК), которые присутствуют во всех органах и тканях человеческого организма и обладают мультилинейным потенциалом дифференцировки. В ряде исследований показано, что способность к хондрогенной дифференцировке МСК из разных тканей различается, однако работы, посвященные данному вопросу, единичны и противоречивы. В соответствии с этическими принципами и технической простотой получения наиболее привлекательными для тканевой инженерии источниками МСК представляются жировая ткань, Вартонов студень пуповины (строма) и пульпа зуба.Целью настоящего исследования было сравнение хондрогенного потенциала МСК, выделенных из жировой ткани, Вартонова студня пуповины и пульпы молочного зуба человека, при культивировании в составе микросфер (пеллет).Материалы и методы. Фенотип первичных культур МСК исследовали методом проточной микроскопии. Хондрогенную дифференцировку проводили при 3D-культивировании в составе микросфер в присутствии TGFβ1 в течение двух недель при стандартных условиях. В качестве положительного контроля использовали хондробласты человека. Жизнеспособность клеток определяли методом флуоресцентного окрашивания. Морфологическое исследование проводили с использованием гистологических и иммуногистохимических методов окрашивания.Результаты. Культуры МСК из всех источников обладали сходным фенотипом CD29+, CD34–, CD44+, CD49b+, CD45–, CD73+, CD90+, HLADR. В составе микросфер визуализировались лишь единичные мертвые клетки в массе живых. Значительная продукция внеклеточного матрикса (ВКМ) отмечалась в микросферах из хондробластов и жировой ткани, тогда как в микросферах из МСК пульпы зуба и стромы пуповины выраженной наработки ВКМ не обнаружили. Среди МСК наибольшую наработку коллагена и гликозаминогликанов (ГАГ) в ВКМ наблюдали в микросферах из МСК жировой ткани, а наименьшую – в микросферах из МСК пульпы зуба.Заключение. Гистологический анализ всех микросфер через 14 суток культивирования в хондрогенной среде выявил признаки дифференцировки в хондрогенном направлении, прогрессивное увеличение продуцируемого клетками ВКМ и присутствие в нем общего коллагена и ГАГ. Из всех исследованных МСК наибольшим хондрогенным потенциалом in vitro (интенсивность наработки компонентов ВКМ) обладают МСК жировой ткани

The comparative study of chondrogenic differentiation of mesenchymal stromal cells allocated from different sources

Introduction. As an alternative to autochondral transplantation, variants of chondrocyte replacement with mesenchymal stromal cells (MSCs) were considered, since these cells are present in all organs and tissues of human body and possess multilinear potential for differentiation. A number of studies demonstrate that the ability for chondrogenic differentiation of MSCs from different tissues varies, however, those studies are few and controversial. In accordance with the ethical principles and the technical ease of obtaining, adipose tissue, Wharton’s jelly (stroma) and dental pulp are the most attractive sources of MSCs for tissue engineering.Aim: to compare the chondrogenic potential of MSCs allocated from adipose tissue, Wharton’s jelly of the umbilical cord, and human deciduous teeth pulp cultured in the composition of microspheres (pellets).Materials and methods. The phenotype of primary cultures of MSCs was studied via flow microscopy. Chondrogenic differentiation was performed with 3D-culture in microspheres in the presence of TGFβ1 for two weeks under standard conditions. Human chondroblasts were used as a positive control. Cell viability was determined by fluorescent staining. Morphological study was performed using histological and immunohistochemical staining methods.Results. Cultures of MSCs from all sources had similar phenotypes CD29+, CD34–, CD44+, CD49b+, CD45–, CD73+, CD90+, HLADR. In the mass of living cells in microspheres only singular dead cells were visualized. A significant production of extracellular matrix (ECM) was observed in the chondroblast and adipose tissue microspheres, whereas in the microspheres of dental pulp and umbilical cord stroma derived MSCs, no significant ECM was detected. Among the MSCs, the highest production of collagen and glycosaminoglycans (GAG) in the ECM was observed in the microspheres of adipose tissue-derived MSCs, and the lowest production – in the microspheres of dental pulp-derived MSCs. Conclusion. A histological analysis of all microspheres after 14 days of culturing in the chondrogenic medium revealed the signs of differentiation in the chondrogenic direction, a progressive increase of the ECM produced by cells, and the presence of total collagen and GAG in it. Of all the MSCs studied, the greatest chondrogenic potential in vitro (the intensity of development of ECM components) is possessed by the adipose tissue-derived MSCs

Интеграция цифровых технологий в профессионально-ориентированное обучение чтению студентов инженерных направлений

Currently, the most crucial social order is to prepare specialists with a number of professional and general cultural competencies. Taking into account that reading is believed to be both a part of a basic literacy and an important component of engineering profile student competences, it is essential to plan teaching reading in the university curriculum. The paper examines the distinguishing features of teaching reading in a foreign language to non-linguistic students in the digital age. The authors present a combination of the traditional reading techniques/strategies and the new ones as an effective means of teaching reading since the digital age has changed the characteristics and the notion of reading. A comprehensive theoretical background of teaching reading is provided. Apart from reading models (bottom-up, top down), types (synthetic, analytical, independent, guided, study, communicative, extensive, intensive) and strategies (scanning, skimming; inferring, monitoring or clarifying, searching and selecting, visualizing and organizing, questioning, SQ3R/SQRRR), special attention is given to the characteristics of the professional texts for students and the types of text tasks. In addition, the authors use the basic principles of language education in the framework of digital technologies in teaching readingВ настоящее время актуальна задача подготовки специалистов, обладающих профессиональными и общекультурными компетенциями. Учитывая, что чтение является базовым навыком функциональной грамотности, а также важным компонентом формируемых компетенций у студентов инженерных направлений, подчеркивается необходимость тщательного планирования обучения чтению в вузе. В статье рассмотрены особенности обучения чтению на иностранном языке студентов нелингвистических специальностей в эпоху цифровых технологий. Авторы представляют совокупность современных и традиционных методов/стратегий чтения в качестве эффективного средства обучения, следуя изменениям в его характеристиках и понятиях в эпоху цифровизации. Дана общая теоретическая основа обучения чтению. Особое внимание уделяется как моделям (снизу вверх, сверху вниз), типам (синтетическое, аналитическое, независимое, управляемое, учебное, коммуникативное, ознакомительное, изучающее), стратегиям (сканирование, беглый просмотр, вывод, мониторинг или уточнение, поиск и отбор, визуализация и организация, чтение с вопросами, SQ3R /SQRRR (Оценить – Задать вопросы – Прочитать – Запомнить – Подвести итоги) чтения, так и специфике текстов профессиональной направленности, типам текстовых заданий. Кроме того, авторы используют основные принципы языкового образования в рамках внедрения цифровых технологий обучения чтени