The bone morphogenetic proteins (BMPs) are cytokines belonging to the transforming growth factor beta (TGF-b) family. They are involved in a plethora of biological phenomena, including osteoblast differentiation and bone formation, key events of the titanium (Ti) implant osseointegration. It is well known that Ti surfaces with nanotopography favor matrix mineralization but the cellular mechanisms implicated in this process are not entirely understood. In this context, we hypothesized that endogenous BMPs, specifically the osteogenic BMP-2/4, have important role in the osteoblast and nanostructured Ti surface interactions. Firstly, MC3T3-E1 pre-osteoblast cell line will be cultured and the following aspects will be evaluated: (1) gene and protein expression of BMP-2/4 in cultures grown on polystyrene by Real-time PCR and ELISA; (2) the efficacy of the BMPR1A, Smad-4, Smad-6, Smurf-1 and Smurf-2 silencers (shRNA) to silence the expression of proteins involved in BMP-2/4 signaling pathways in cultures grown on polystyrene by Western blot; and (3) the shRNA effects on the BMP-2/4 signaling pathways and on the osteoblast phenotype expression in cultures grown on polystyrene by PCR-array and Western blot. To test our hypothesis, it will be evaluated: (1) the effect of Ti with nanotopography, compared to control Ti, on the BMP-2/4 synthesis in cultures grown on both surfaces by Real-time PCR and ELISA; and (2) the shRNA effects on the BMP-2/4 signaling pathways and on the osteoblast phenotype expression in cultures grown on Ti with nanotopography compared to control Ti. The outcomes of this study could contribute to clarify the intracellular mechanisms involved in osteoblast responses to Ti and consequently novel strategies using BMPs and nonotopography could be developed in order to favor the process of osseointegration.
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