In Vitro Osteoclastogenesis: Proteomic Gel-free Analysis

Periapical bone resorption is a key factor for endodontic treatment success. Oral-related bone resorptions have a direct relation to oral immune response in a similar way of other systemic bone resorptions processes. Objective: To assess the global proteomic of osteoclast precursors RAW264.7 cell line (RAW) stimulated or not with receptor activator nuclear factor Kappa B ligand (RANKL), an essential cytokine for osteoclastogenesis process. Method: RAW cells were stimulated in the presence or absence of recombinant (r) RANKL for 7 days in order to to assess cell viability, number of differentiated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells, nitric oxide (NO) production and further proteomic analysis. The proteomic analyses were based on nanoscale ultra performace liquid chromatography mass spectrometry with an independent data method acquisition (NanoUPLC-MS^E). Result: Both groups did not present differences in cell viability. The RANKL-stimulated cell culture presented higher number of TRAP-positive multinucleated cells comparing to no-stimulated cultures (p<0,05, by Student T test). Regarding to the NO production, the highest production was observed in day 3 cultures and the highest production was observed in RANKL-stimulated RAW cells culture (p<0.05, by Student T test). The proteomic analysis allowed the identification of 124 common proteins and 86 differential expressed proteins. Among these proteins, 45 proteins are present only in Raw cells culture, while other 41 proteins are found only in RANKL-stimulated Raw cells. The identified proteins of both groups could be strictly related to proliferation, metabolism and cell signaling. In addition, there were some proteins related to immune response, cytoskeleton dynamic and organization and cell motility. Conclusion: Proteins identified in this work may help to define down-regulation biomarkers to periapical bone resorption. These markers could shed some light over new therapies and medications, which may be developed in future, enabling a higher success rate for curative therapies.