TESTING THE INFLUENCE OF GROWTH FACTORS, EXTRA-CELLULAR MATRIX COMPONENTS, PLANT EXTRACTS AND ORGANIC DETERGENTS, ON THE OSTEOBLAST AND OSTEOCLAST IN VITRO-DIFFERENTIATION DIRECTION OF HUMAN AND MOUSE CELL PROGENITORS FROM DIFFERENT SOURCES

International Journal of Pharmaceutical Research and Innovation, Vol. 8, 2015, 23-28

Iskra Sainova, Ilina Valkova, Bistra Alexieva, Elena Nikolova

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ABSTRACT

Techniques for differentiation of progenitor cells from different sources of human and mouse origin, in osteoblast and osteoclast directions, were developed and investigated. The used precursor cells were from two different sources of the used objects: from human bone marrow mesenchymal stromal/stem cells (MSCs) and from Balb/c mouse embryonic fibroblasts 3T3, respectively. Normal human stromal/stem cells were derived from bone marrow material. Osteoblast derivation from the human bone marrow progenitors was achieved by pre-cultivation of separate sub-populations of nucleated cell precursors in the presence of Ascorbic acid (Vitamin C), Dexamethazone and β-Glycerophosphate. Similarly, osteoclasts with human origin were derived from other sub-populations of the same precursors, by pre-cultivation in the presence of Macrophage-Colonia Stimulation Factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). After formation of confluent monolayer from mouse embryonic 3T3 cells, separate sub-populations from them were pre-incubated in cultural fluid, supplemented by cultivation of mouse malignant myeloma cells in it, as well as with fruit extract from the medical plant Aronia melanocarpa, subsequent freezing of the cell suspensions at -80oC for 2-4 weeks in the presence of  respective volume from the cryo-protector Dymethylsulfoxide (DMSO) and thawing, appearance of multi-nuclear osteoclast-like cells was noted. In additions of cultural fluid from the so derived cells to de novo-formed confluent monolayer of 3T3 fibroblasts, osteoblast-like cell characteristics were observed, as formation of dense dark-staining formations. The results obtained indicated the influence of feeder, incubation and extra-cellular matrix factors, but also of the organic detergents, on the cell differentiation direction, which, on the other hand, could explain indirect negative effects of organic detergents on the process of osteogenesis. Furthermore, a role of DMSO and other organic detergents as stimulators of the fusion process, was proposed.

Keywords: osteoblast and osteoclast cell differentiation, cell progenitors, growth factors/cytokins, fruit extracts, organic detergents.