Leukotriene B(4) loaded in microspheres regulate the expression of genes related to odontoblastic differentiation and biomineralization by dental pulp stem cells

BACKGROUND: Leukotriene B(4) (LTB(4)) is a potent lipid mediator that stimulate the immune response. Because dental pulp inflammation and dentin repair are intrinsically related responses, the aim of this research was to investigate the potential of LTB(4) in inducing differentiation of dental pulp stem cells. METHODS: Microspheres (MS) loaded with LTB(4) were prepared using an oil emulsion solvent extraction evaporation process and sterility, characterization, efficiency of LTB(4) encapsulation and in vitro LTB(4) release assay were investigated. Mouse dental pulp stem cells (OD-21) were stimulated with soluble LTB(4) or MS loaded with LTB(4) (0.01 and 0.1 μM). Cytotoxicity and cell viability was determined by lactate dehydrogenase and methylthiazol tetrazolium assays. Gene expression were measured by quantitative reverse transcription polymerase chain reaction after 3, 6, 24, 48 and 72 h. Mineralized nodule formation was assessed after 28 days of OD-21 cell stimulation with LTB(4) in mineralized media or not. Groups were compared using one-way ANOVA test followed by Dunnett’s post-test (α = 0.05). RESULTS: Treatment with LTB(4) or MS loaded with LTB(4) (0.01 and 0.1 µm-μM) were not cytotoxic to OD-21 cells. Treatment with LTB(4) modulated the expression of the Ibsp (integrin binding sialoprotein) and Runx2 (runt-related transcription factor 2) genes differently depending on the experimental period analyzed. Interestingly LTB(4) loaded in microspheres (0.1 μM) allowed long term dental pulp cell differentiation and biomineralization. CONCLUSION: LTB(4), soluble or loaded in MS, were not cytotoxic and modulated the expression of the Ibsp and Runx2 genes in cultured OD-21 cells. When LTB(4) was incorporated into MS, odontoblast differentiation and mineralization was induced in long term culture.