Prevention of osteonecrosis of jaws induced by medicines with the use of photodynamic therapy. experimental study in vitro and in vivo

Abstract

Bisphosphonates are anti-resorptive drugs used in the treatment of skeletal disorders, such as osteoporosis, but their prolonged use may induce osteonecrosis of the jaw (MRONJ), which represents a challenging treatment for dentistry. The aim of this project will be to evaluate the preventive effect of therapies on MRONJ during the alveolar repair process in rats. For this, 360 wistar rats will be used, which will be treated with 04 applications of 0.035 mg / kg of zoledronic acid (ZOE) through the caudal vein with 15 days intervals between applications and subsequently have their right lower molars removed. After extraction, the animals will continue treatment with ZOE in the same protocol until euthanasia. The animals will be divided into seven groups according to the treatment of the dental socket: GS (Sham - ZOE will apply), GC (blood clot only), G² (²-tricalcium phosphate), GD (10% doxycycline), GP (photo-dynamic therapy), GD² (10% doxycycline + ²-tricalcium phosphate), GP² (photo-dynamic therapy + ²-tricalcium phosphate), GPD (photo-dynamic therapy + 10% doxycycline), GPD² (photo-dynamic therapy + 10% doxycycline + ²-tricalcium phosphate). For in vitro analysis by primary cell culture to determine the biological parameters will be used 72 animals, and analyzes will be performed by 1) Cell Proliferation Evaluation; 2) Analysis of Total Protein Content; 3) Real-Time RT-PCR Gene Expression Analysis (Runx2, ²-catenin, osterix, alkaline phosphatase, osteocalcin, collagen type I, rank, rankl, osteopontin and bone sialoprotein); 4) Biological Mineralization Detection and Quantification; 5) Immunofluorescence Analysis of Bone Proteins; 6) Analysis of Node-like Mineralized Bone Formation. For in vivo analysis, in the calcified cuts will be used 72 animals, where after 36 days from the date of extraction, the animals will be euthanized for evaluation by 1) application of fluorochromes (calcein and alizarin) followed by confocal laser microscopy analysis. and 2) microtomographic analysis. For in vivo analysis, the decalcified sections will be used 216 animals, where after 07, 14 and 38 days from the date of extraction, the animals will be euthanized for evaluation by 1) histometric and histomorphometric analysis 2) immunohistochemistry through expression osteocalcin (OC) protein, nuclear factor kappa B receptor activating receptor (RANKL), tartrate-resistant acid phosphatase (TRAP), 3) RT-PCR molecular analysis with osteoprotegerin gene expression (OPG), alkaline phosphatase (ALP), osteocalcin (OC), RANKL 4) birefringence analysis with picrosirius-Red staining. (AU)