Phenotypic Transition of Periodontitis-derived Macrophages

Success of bone grafts depends on their ability to induce favorable cellular responses. Accordingly, bone regeneration is thought to depend on the ability of macrophages to undergo transition from M1 to M2 phenotype. Macrophages from grade C periodontitis subjects are hyper-inflammatory, nonetheless it is unknown whether these cells undergo efficient M1-M2 transition. The aim of this study is to evaluate the ability of macrophages from grade C periodontitis to undergo phenotypic transitions. In brief, macrophages from generalized grade C periodontitis (n=10), generalized grade B periodontitis (n=10) and periodontally healthy (n=10) subjects will be exposed to M1- and M2-polarizing stimuli and in vitro kinetics of macrophage phenotype switch will be assessed using flow cytometry analysis of the cells and multiplex assays of conditioned media. Data will be analyzed by one-way ANOVA followed by post-hoc Tukey's test (α = 0.05). Preliminary experiments were conducted to determine ideal experimental conditions for peripheral blood mononuclear cell (PBMC) isolation, CD14+ isolation, differentiation of PMBCs into macrophages and polarization of M0 macrophages into M1 and M2 macrophages. CD14+ negative magnetic isolation and an adherence protocol were necessary to obtain predictable macrophage differentiation. Ideal methods for PBMC isolation, PBMC differentiation into macrophages, and M0 macrophage polarization into M1 and M2 macrophages were established. Keywords: periodontitis, macrophages, inflammation, healing