vtioВестник трансплантологии и искусственных органовRussian Journal of Transplantology and Artificial Organs1995-1191Academician V.I.Shumakov National Medical Research Center of Transplantology and Artificial Organs", Ministry of Health of the Russian Federation10.15825/1995-1191-2019-1-101-112vtio-996Research ArticleРегенеративная медицина и клеточные технологииRegenerative Medicine and Cell TechnologiesСравнительное исследование процесса хондрогенной дифференцировки мезенхимальных стромальных клеток, выделенных из разных источниковThe comparative study of chondrogenic differentiation of mesenchymal stromal cells allocated from different sourcesБасокЮ. Б.BasokYu. B.

123182, Москва, ул. Щукинская, д. 1.Тел. (499) 193-86-62. 

1, Shchukinskaya str., Moscow, 123182. Теl. (499) 193-86-62.

bjb2005@mail.ruГригорьевА. М.GrigorievA. M.

Москва

Moscow

КирсановаЛ. А.KirsanovaL. A.

Москва

Moscow

ВахрушевИ. В.VakhrushevI. V.

Москва

Moscow

ЦветковаА. В.TsvetkovaA. V.

Москва

Moscow

ГрядуноваА. А.GryadunovaA. A.

Москва

Moscow

ЯрыгинК. Н.YaryginK. N.

Москва

Moscow

СевастьяновВ. И.SevastianovV. I.

Москва

Moscow

ФГБУ «Национальный медицинский исследовательский центр трансплантологии и искусственных органов имени академика В.И. Шумакова» Минздрава РоссииРоссияV.I. Shumakov National Medical Research Center of Transplantology and Artificial Organs of the Ministry of Healthcare of the Russian FederationRussian FederationФГБНУ «Научно-исследовательский институт биомедицинской химии имени В.Н. Ореховича»; Лаборатория биотехнологических исследований «3Д Биопринтинг Солюшенс»РоссияV.N. Orekhovich Institute of Biomedical Chemistry; Private Institution Laboratory for Biotechnological Research «3D Bioprinting Solutions»Russian FederationФГБНУ «Научно-исследовательский институт биомедицинской химии имени В.Н. Ореховича»РоссияV.N. Orekhovich Institute of Biomedical ChemistryRussian FederationЛаборатория биотехнологических исследований «3Д Биопринтинг Солюшенс»; ФГАОУ ВО «Первый Московский государственный медицинский университет имени И.М. Сеченова» Минздрава России (Сеченовский университет)РоссияPrivate Institution Laboratory for Biotechnological Research «3D Bioprinting Solutions»; I.M. Sechenov First Moscow State Medical University of the Ministry of Healthcare of the Russian Federation (Sechenovsky University)Russian Federation201917052019211101112Copyright © Басок Ю.Б., Григорьев А.М., Кирсанова Л.А., Вахрушев И.В., Цветкова А.В., Грядунова А.А., Ярыгин К.Н., Севастьянов В.И., 20192019Басок Ю.Б., Григорьев А.М., Кирсанова Л.А., Вахрушев И.В., Цветкова А.В., Грядунова А.А., Ярыгин К.Н., Севастьянов В.И.Basok Y.B., Grigoriev A.M., Kirsanova L.A., Vakhrushev I.V., Tsvetkova A.V., Gryadunova A.A., Yarygin K.N., Sevastianov V.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://journal.transpl.ru/vtio/article/view/996Введение

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

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

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

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.

Introduction

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

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

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

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.

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The authors declare that there are no conflicts of interest present.