Evaluation of citokines VEGF, TNF-a, RANKL and OPG in repair of bone defects on skull of diabetic rats treated with demineralized bone matrix

• Grant number: 10/09934-8
• Support Opportunities: Regular Research Grants
• Start date: March 01, 2011
• End date: February 28, 2013
• Field of knowledge: Biological Sciences - Morphology - Histology
• Principal Investigator: Gerson Francisco de Assis
• Grantee: Gerson Francisco de Assis
• Host Institution: Faculdade de Odontologia de Bauru (FOB). Universidade de São Paulo (USP). Bauru , SP, Brazil
• Associated researchers: Bruna Barros Bighetti ; Gustavo Pompermaier Garlet ; Rumio Taga

Abstract

Studies had demonstrated that Diabetes Mellitus helps the bone loss, being common the femurs head and/or lumbar spine osteopenia, as well as osteoporosis, furthermore it shows negative signals also during healing repair, as angiogenesis and blood suplies reduction, severe inflammatory response, collagen synthesis reduction, disorder in mineralization process and a imbalance between bone resorption by osteoclasts and matrix deposition by osteoblasts. In this case, the participation of two signallings molecules has a essential hole: the tumoral necrosis factor (TNF-alfa) that promotes the expretion of activator for ligant kB-nuclear factor (RANKL) by T-lymphocytes, B-lymphocytes, endothelial cells and osteoblasts. The RANKL molecule binds to RANK-receptor located on forerunner cells surface, promoting its diferenciation to osteoclast. To control osteoclastogenesis, osteoprotegerin receptor molecule (OPG) competes with RANK for linkage with RANKL, connecting with RANKL and preventing diferenciation. Finally, the vascular endothelial growth factor (VEGF) promotes angiogenesis, becoming a prerequisite for local recruitment of osteoclast precursor cells and osteoblasts during bone formation and repair. Furthermore, os allogenic bonne grafts (MAOD), from an individual donor of the same species as the receptor but with different genotype, has been an alternative in the treatment of extensive bone defects, due to its osteoconductivity and osteoinductivity. Thus, the current study aims to determine whether the MAOD improves the repair of bone defects in diabetic and normoglycemic rats and changes or not the expression of VEGF, RANKL and OPG and TNF-alfa. To do this, will be used 200 male Wistar rats with 90 days old divided into non diabetics group (n = 100), which will receive a dose of saline solution 0.9% and diabetic group (n = 100), which will be induced diabetes by applying a single intraperitoneal dose of streptozotocin (47 mg / kg). At the end of experimental periods of 0, 7, 14, 21 and 42 days (10 animals in each subgroup / period) the calvarias will be collected and 5 fixed in 10% formalin and subjected to histological processing for morphometric analysis and immunohistochemistry, and 5 will be crushed, a half wrapped in Trizol and frozen for analysis of mRNA by RT-PCR real-time, and the other half just frozen for protein analysis by Western blotting for VEGF, RANKL, OPG and TNF-alfa. Faced with the proposed analysis, we will check: a) the volume of newly formed bone tissue in both conditions (diabetes and normoglycemic) and treatment (untreated and treated with MAOD), b) VEGF protein expression and the pattern of vascularization and ossification, and c ) the number of osteoclastos/mm2 kinetics in both conditions (diabetes and normoglycemic) and treatment (untreated and treated with MAOD) and d) correlate the expression of the protein TNF-alfa, RANKL and OPG with the matrix reabsorption. With the results of this analysis we will check if MAOD improves the repair of bone defects in diabetic and normoglycemic rats and if changes or not the expression of VEGF, RANKL and OPG and TNF-alfa. (AU)