Bone marrow and stem cells in periodontal regeneration: microtomographic, histomorphometry, immunohistochemistry and immunoenzymatic analysis in periodontal fenestration defects in rats

Abstract

The objective of this study is to evaluate the regenerative potential of bone marrow concentrated aspirate (BMCA) and cultured mesenchymal stem cells derived from bone marrow (MSCBM) in the treatment of surgically created periodontal fenestration defects in rats. MSCBM cultured and isolated CD105+, CD34- and CD45- will be obtained from 2 isogenic mice by the magnetic cell separation. To assess the purity of the separated cells the flow cytometry and immunostaining for STRO-1 will be performed. Samples will be subjected to adipogenic and osteogenic differentiation in order to check through genetic analysis, Von Kossa and Adipogenic assays the differentiation capacity of separated MSCBM. Periodontal fenestration defects will be created bilaterally in the mandible of other 168 mice. The animals will be randomly divided into 7 groups: 1) C Group (Control): the defect will be filled with blood clot, 2) PGA Group: the defect will be filled with Propylene Glycol Alginate, 3) PEMD Group: the defect will be filled with Protein Enamel Matrix Derived, 4) BMCA-PGA Group, 5) BMCA-PEMD Group 6) PGA-MSCBM Group and 7) MSCBM-PEMD Group. The animals will be divided into 3 subgroups for euthanasia at 15, 30 and 60 days postoperatively. In one hemi-mandible will be assessed the volume and linear measurements of newly formed tissue by the microtomographic analyse, the type and quality of newly formed tissue by the histologic and histomorfometric analyses and evaluate the presence of osteocalcin, osteopontin, osteonectin, RANK-L, RUNX-2, TRAP and bone sialoprotein by the imunohistoquimic analyse. On the other hemi-arch will immunoassay to determine the levels of IL-6, TNF-a, MCP-1, IL-10, IL-1ß, TGF-b, RANKL, OPG, IL-8 and M-CSF. The data will be statistically analyzed, adopting a significance level of 5%. (AU)