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Culturing of stromal cells derived from rat bone marrow in different origin I type collagen

PDF, Volume IV, №4, 2009

Barmasheva A.A.¹, Sharutina I.A.², Nikolaenko N.S.³, Kukhareva L.V.³, Shamolina I.I.², Pinaev G.P.³

1. The Saint Petersburg I.P. Pavlov State Medical University, Saint Petersburg, Russia
2. The Saint Petersburg State University of Technology and Design, Saint Petersburg, Russia
3. The RAS Institute of Cytology, Saint Petersburg, Russia

Keywords:
bone marrow stromal cells, type I collagen, spreading, cytoskeleton, proliferation, differentiation

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Type I collagen is widely used in medicine and biotechnology owing to its ready availability and good biological properties. The aim of this study was to investigate proliferation (proliferation index), spreading (actin cytoskeleton, spreading square) and osteogenic differentiation of the rat bone marrow stromal cells (BMSCs) on the different molecular and fibrillar collagen substrates on the surface and during three-dimensional cultivation in the collagen gels. Collagen samples (1, 2, 3) were obtained from the skin of the sheep and young bull by alkaline extraction. Control collagen (4) was obtained by acid extraction from rat tail tendons. Fibrillar collagen on the surface was prepared by subsequent threefold application of the molecular collagen layers on the substrate. The capacity to form gels of the collagen samples in neutral medium at 37^oC was examined. BMSCs of the outbread rats were obtained by centrifugation on the histopaque gradient with 1,077 g\ml density and were cultured in the growth medium. Actin cytoskeleton of the spreaded cells was stained with rhodamine-phalloidin. Distribution of the early osteogenic differentiation marker — the alcaline phosphatase — was elicited histochemically by staining of the cells with the standard solution containing tetrazolium blue. In the course of the study was determined that not only control collagen obtained from rat tails (4), but one collagen sample obtained from the sheep skin (1) has the capacity to form gel. Hereafter we compared cell actions on the fibrillar (gel-forming) (1 and 4) and molecular (not gel-forming) (2 and 3) collagen samples. BMSCs had maximal spreading square, proliferation index and alcaline phosphatase activity on the fibrillar collagens (1 and 4). Both collagens formed stable gels in which cells kept osteogenic differentiation capacity until two weeks. Gel-forming sample of the type I collagen obtained from the sheep skin can be recommended as a scaffold for BMSCs transplantation.

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