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Association of the phytoestrogen Vitex agnus-castus and the conditioned medium of M1 polarized macrophages in vivo and in vitro models to study the bone metabolism of ovariectomized rats.

Grant number: 22/13865-9
Support Opportunities:Scholarships in Brazil - Master
Effective date (Start): June 01, 2023
Effective date (End): May 31, 2025
Field of knowledge:Health Sciences - Dentistry
Principal Investigator:Karina Fittipaldi Bombonato Prado
Grantee:Sayuri Poli Suguimoto
Host Institution: Faculdade de Odontologia de Ribeirão Preto (FORP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil


Recent data suggest that macrophage polarization in pro-inflammatory (M1) and anti-inflamatory states (M2) may interfere in the osteoblast differentiation and osteogenesis modulation in critical bone defects in the presence of osteoporosis, where chronic inflammation is a relevant factor. The utilization of natural antioxidant and anti-inflammatory substances as an alternative to hormonal replacement ther-apy and other utilized drugs could avoid side effects and delays in bone neoformation, mainly in post-menopausal women. Thus, the objective of this project will be to evaluate the bone repair and the oste-oblastic functional activity of ovariectomized rats gavaged with the phytoestrogen Vitex agnus-castus, in association with M1 macrophages conditioned medium. For in vivo experiments, Wistar Hannover female rats will be ovariectomized and will receive critical bone 5 mm defects in calvaria. Immediately after surgery, the extract of Vitex agnus-castus will be given by gavage every other day for 90 days, until euthanasia, as well as the application of conditioned medium of macrophages polarized in M1, applied in the region of the defect once a week until euthanasia. The rats will be divided into 8 groups: 1 - SHAM and 2 - OVX (water administration), 3 - SHAMmc e 4- OVXmc (water administration + inoculation of conditioned medium of macrophage) 5- SHAMvx and 6- OVXvx (Vitex agnus-castus administration), 7 - SHAMvx/ad and 8 - OVXvx/ad (Vitex agnus-castus administration + inoculation of distilled water), 9 - SHAMvx/mc and 10 - OVXvx/mc (Vitex agnus-castus administration + inoculation of conditioned medium of macrophage). After that, samples from the calvaria will be coll-ected for histological processing, and samples from the femurs will be collected for the isolation of bone marrow cells. The qualitative histological analysis of the histological plates will be performed with HE images. The quantitative histological analysis of the newly formed trabecular bone will be performed using Im-age J software. The culture of bone marrow mesenchymal cells obtained from the femurs will be carried out in bottles in an osteogenic culture medium until subconfluence and plated at a concentration of 2 x 104 cells per well in culture plates (n=5). The experimental groups will be divided into 1 - Osteoblastic cells + osteogenic medium, 2 - Osteoblastic cells + macrophage conditioned medium, 3 - Osteoblastic cells + Vitex agnus-castus, and 4 - Osteoblastic cells + macrophage conditioned medium + Vitex agnus-castus. After the experimental times, the following parameters will be evaluated: cell proliferation, in situ detection of ALP, and detection and quantification of mineralized matrix. The data obtained will be analyzed by statistical programs consistent with significance p<0.05.

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