In vitro evaluation of cell adhesion, proliferations and protein expression by means of immunolocalization of osteoblastic cells MC3T3-E1 cultured with conditioned medium of raw 264.7 cell line macrophages
Osteogenesis is achieved not only by osteoblastic cells but also as a result from cooperation with several systems such as immune system. Macrophages may secrete several signaling molecules wich may affect in a good or bad way the osteogenic capacity of osteoblasts. Thus, the objective of this project will be to evaluate the influence of a medium conditioned with RAW264.7 macrophages polarized for M1 (by means of LPS) in the culture of MC3T3-E1 osteoblastic cells. After the storage of conditioned medium, osteoblastic cells will be cultured in flasks in the osteogenic medium until subconfluence and seeded at a concentration of 2 x 104 cells per well in culture plates (n = 5) on glass slides adequate for cell adhesion and proliferation. Experimental groups will be divided in control cells (C) and treated with 50% of the conditioned medium (MC). After the experimental period, the following parameters will be evaluated: cell adhesion proliferation, as well as expression of alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteopontin (OPN) by means of immunolocalization. The results will be analyzed by statistical programs consistent with significance p d 0,05.
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