Results from our research group have shown that the titanium (Ti) surface with nanotopography induces osteoblast differentiation by regulating the endogenous production of bone morphogenetic protein 2 (BMP-2) in a process, at least in part, mediated by BMPR1A receptor. Additionally, the involvement of the canonical Wingless-Type mouse mammary tumor virus [MMTV] Integration Site (Wnt) and non-canonical (Wnt/Ca2+) signaling pathways in the response of osteoblasts to Ti surface modifications has been investigated. As both pathways cooperatively regulate bone formation, the aim of this study will be to evaluate the role of the interaction of the Wnt and BMP signaling pathways in the osseoinductive effect of the Ti with nanotopography. MC3T3-E1 osteoblastic cells grown on polystyrene will be transfected with shRNA for target genes of the canonical Wnt and non-canonical Wnt/Ca2+ pathways, selected based on their modulation by nanotopography. Then, the effect of the silencing of both shRNAs (canonical Wnt and non-canonical Wnt/Ca2+ pathways) on osteoblast differentiation will be evaluated as follows: 1) the gene expression of RUNX2, alkaline phosphatase (ALP) and osteocalcin (OC); 2) the protein expression of RUNX2 and ALP and 3) the ALP activity. The shRNA that induced a more pronounced negative effect on osteoblast differentiation will be selected to be used in combination with shRNA for BMPR1A receptor to evaluate the role of the interaction of Wnt and BMP signaling pathways on the osseoinductive effect of Ti with nanotopography. We will evaluate the effect of silencing of the selected shRNA (canonical Wnt or non-canonical Wnt/Ca2+), the shRNA for BMPR1A receptor and the combination of both on the osseoinductive potential of Ti with nanotopography. Cells will be cultured on Ti surface with nanotopography and on machined Ti surface (control) and the same parameters of osteoblast differentiation described above will be evaluated. The results of this study may contribute to the understanding of the intracellular mechanisms involved in the osteoblast response to Ti surfaces and to the development of new strategies involving cell signaling and surface nanotopography that could favor the events related to the osseointegration process.
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