| Grant number: | 20/12726-0 |
| Support Opportunities: | Regular Research Grants |
| Start date: | March 01, 2022 |
| End date: | October 31, 2024 |
| Field of knowledge: | Health Sciences - Dentistry |
| Principal Investigator: | Carla Renata Sipert |
| Grantee: | Carla Renata Sipert |
| Host Institution: | Faculdade de Odontologia (FO). Universidade de São Paulo (USP). São Paulo , SP, Brazil |
| City of the host institution: | São Paulo |
| Associated researchers: | Cristina Cunha Villar ; Fernando Neves Nogueira ; Maria Helena Raposo Fernandes ; Marinella Holzhausen Caldeira ; Rui de Albuquerque Carvalho |
Abstract
Mineralized tissue resorption is a biological event present in challenging oral pathologies such as periodontal diseases, apical periodontitis and external root resorption. The role of mesenchymal cells at bone metabolism modulation is described, but not the role of apical papilla cells under this context, specially by means of osteoprotegerin (OPG) effect. The aim of this study is to investigate the role of OPG produced by stem cells of the apical papilla (SCAP) at apoptosis, migration and differentiation of monocytes in vitro. Primary culture of SCAP will be established, and the cells will be subjected to OPG silencing by liposomal transfection (SCAP-OPG KD or SCAP-scr KD). The supernatant will be collected for silencing validation by means of OPG quantification as well as other factors involved with apoptosis such as TRAIL and Interleukin(IL)-33 or with cell migration such as CCL2. Besides that, receptor activator of nuclear factor kappa B ligand (RANKL) will be detected. Based on these findings, conditioned media will be prepared and stored. Peripheral blood monocytes will be isolated from healthy volunteers and cultivated. These cells will be treated with SCAP-OPG KD or SCAP-scr KD supernatants and will be investigated upon cell viability and apoptosis at the presence or not of TRAIL and IL-33 neutralizing antibodies. For cell migration, the conditioned media will be used together with CCL2 neutralizing antibody. Finally, monocytes will be induced for osteoclast differentiation by adding M-CSF and RANKL at the presence of SCAP conditioned medium or control medium for 21 days. Osteoclast differentiation will be assessed by means of actin rings analuysis, detection of tartrate resistant acid phosphatase (TRAP) and cathepsin K, TRAP activity and glucose consumption and lactate production. SCAP will be immunostained for membrane RANKL and then cocultured with CD14+ monocytes for 14 days. TRAP, cathepsin K and RANKL will be assessed by immunofluorescence as well as TRAP activity. Data will be statistically analyzed based on normality distribution standard. Significant p values will be set at 5%. (AU)
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