Evaluation of biomineralization responses by Rubus coreanus in estrogen deficiency: a translational study

Abstract

This project will investigate the local action of Rubus coreanus through the functionalization of the surface of the implants to be installed. This project will be carried out in 2 stages. In the first stage, the characterization of the surface of implants functionalized by drugs will be carried out, based on physical chemical and biological tests in cell cultures. For cell culture, undifferentiated mesenchymal cells, isolated from rat femurs, will be used. Cell differentiation and activity will be assessed. Subsequently, the second stage will be performed where the experiments will be performed in vivo. For this purpose, 40 young adult female Wistar rats will be used, with 16 rats for the first euthanasia period (14 days), and 24 rats for the second euthanasia period (28 days). The experimental groups will be: SHAM CONV, SHAM RC, OVX CONV, OVX RC, and OVX animals will be submitted to bilateral ovariectomy, while SHAM animals will be submitted to fictitious bilateral ovariectomy surgery. After 30 days, all animals will undergo surgery to install the implants in the tibial metaphyses. The implants will have the surfaces treated with Rubus coreanus, and there will be the negative control that will receive conventional implants. The euthanasia of animals from the first period of euthanasia will occur 14 days after the installation of the implants and the tibiae will be used for the following analyzes: biomechanics (counter-torque) and real-time PCR (expression of the Runx-2, ALP, IBSP genes, Wnt3a, OPG and RANKL), while on the contralateral side immunohistochemistry will be performed (immunostaining of Runx-2, ALP, IBSP, Wnt3a, OPG and RANKL proteins). For animals in the second period of euthanasia, fluorochromes (20mg / kg) will be administered, with calcein being 14 days after implant placement and alizarin 24 days after implant placement, and euthanasia will be performed 28 days after installation of implants and tibiae will be used for the following analyzes: biomechanics (counter-torque) and real-time PCR (expression of Runx-2, ALP, IBSP, Wnt3a, OPG and RANKL genes), while on the contralateral side computed microtomography for morphometric evaluation of the bone formed around the installed implants. Subsequently, these same samples will be subjected to laboratory processing for calcified cuts, for later analysis by confocal microscopy (peri-implant bone dynamics). Quantitative data will be submitted to the homoscedasticity test for the selection of the appropriate statistical test (parametric or non-parametric), with a significance level of 5%. (AU)