Abstract
The article presents data on the cultivation and characteristics of mesenchymal stem cells (MSC) isolated from the bone marrow of patients with dysplastic coxarthrosis. Several morphological phenotypes were found in the fraction of adhesive MSC: spindle-shaped elongated cells, large flattened cells, and thin stellate cells in both samples of bone marrow. Immunophenotypic analysis showed that cells express surface antigens (CD90, CD73, CD105, CD45 and CD34), which are characteristic for typical stem cells. It was shown that the use of a new growth medium containing no components of animal origin for the cultivation of human MSC allowed to achieve confluence of the cell culture on the 16th-8th day of incubation without delaying the proliferative activity of the cells and without loss of ability to differentiate into chondro- and osteogenic types of tissues. Multipotency of MSC was confirmed by osteogenic and chondrogenic differentiation of cells, during prolonged cultivation of MSCs in induction media in vitro . The differentiation of MSC into osteoblasts was confirmed by immunocytochemical staining for alkaline phosphatase and alizarin red S. Specific differentiation of MSC in chondrogenic type was revealed by staining of cartilage deposits with alcian blue. For the first time, such characteristics of human MSC as: mitotic index, trajectory of cells migration and average speed of migration on culture plastics were determined. The mitotic index of actively proliferating MSC was from 2.7 to 3.4 % of the total cell number. The moving activity (speed of cell migration) was 38-42 μm/h. Thus, bone marrow aspirate from patients with orthopedic pathology is the source of stem cells that meet all the criteria for MSC as determined by the International Society of Cellular Therapy and can be used in regenerative therapy of bone and cartilage.
Highlights
Одной из главных проблем травматологии и ортопедии остается замещение дефектов костной ткани опорно-двигательного аппарата
Обеспечивая новое физиологическое микроокружение в области трансплантации in vivo [8]
Bone marrow aspirate from patients with orthopedic pathology is the source of stem cells that meet all the criteria for mesenchymal stem cells (MSC) as determined by the International Society of Cellular Therapy and can be used in regenerative therapy of bone and cartilage
Summary
МСК человека были выделены из аспиратов костного мозга резецированных головок бедренных костей пациентов с диспластическим коксартрозом при операции первичного эндопротезирования тазобедренных суставов в Новосибирском НИИ травматологии и ортопедии им. Суспензию клеток костного мозга разводили в соотношении 1 : 2 0,9%-м NaCl, наслаивали на градиент плотности PANcoll (PAN biotech, Германия) (d = 1,077 г/мл) и центрифугировали при 1300 g в течение 15 мин [1, 2]. Ядросодержащие клетки переносили в отдельную пробирку, отмывали от PANcoll центрифугированием при 1500 g в течение 15 мин в бессывороточной среде DMEM с добавлением 100 Ед/мл пенициллина, 100 мкг/мл стрептомицина и 2,5 мкг/мл фунгизона. Культивирование клеток с нулевого пассажа проводили в специальной бессывороточной ростовой среде PowerStem MSC1 (PAN biotech), содержащей 100 Ед/мл пенициллина, 100 мкг/мл стрептомицина и 2,5 мг/мл фунгизона, при 37 °С в СО2-инкубаторе (5 % концентрации СО2). Для визуализации минеральных отложений внеклеточного матрикса клетки после 21 суток инкубирования в индукционной среде дважды промывали в PBS (без Са++ и Mg++), наслаивали 10%-й формалин и фиксировали в течение 15 мин при комнатной температуре. Клетки обоих типов ткани анализировали и фотографировали с помощью микроскопа Axio Observer Z1 (Carl Zeiss, Германия) и программного обеспечения ZEN 2012 (blue edition)
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