Abstract
BackgroundLeukotriene B4 (LTB4) 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 LTB4 in inducing differentiation of dental pulp stem cells.MethodsMicrospheres (MS) loaded with LTB4 were prepared using an oil emulsion solvent extraction evaporation process and sterility, characterization, efficiency of LTB4 encapsulation and in vitro LTB4 release assay were investigated. Mouse dental pulp stem cells (OD-21) were stimulated with soluble LTB4 or MS loaded with LTB4 (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 LTB4 in mineralized media or not. Groups were compared using one-way ANOVA test followed by Dunnett’s post-test (α = 0.05).ResultsTreatment with LTB4 or MS loaded with LTB4 (0.01 and 0.1 µm-μM) were not cytotoxic to OD-21 cells. Treatment with LTB4 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 LTB4 loaded in microspheres (0.1 μM) allowed long term dental pulp cell differentiation and biomineralization.ConclusionLTB4, soluble or loaded in MS, were not cytotoxic and modulated the expression of the Ibsp and Runx2 genes in cultured OD-21 cells. When LTB4 was incorporated into MS, odontoblast differentiation and mineralization was induced in long term culture.
Highlights
Pulp and dentin are closely related tissues, being assembled as a single unit, the dentin-pulp complex, which is a strategic and dynamic barrier in face of injuries suffered by teeth, being caries the most common cause of injury to this complex [1, 2]
In the presence of 5-Lipoxygenase activating protein (FLAP) (5-lipoxygenase activating protein), a nuclear protein associated with the membrane, the enzyme 5-LO is activated and oxidizes arachidonic acid, converting it to 5S-hydroxyperoeicosatetraenoic acid (5S-HpETE), which is further reduced by the enzyme peroxidase to 5S acid-hydroxyieicositetraenoic (5S-HETE) or is converted into Leukotriene A4 (LTA4), which, by the action of LTA4 hydrolase, results in Leukotriene B4 (LTB4) production [11]
Analysis of LTB4 release showed a burst release from MS at 6 h, when approximately 20% of the mediator was detected in the medium
Summary
Pulp and dentin are closely related tissues, being assembled as a single unit, the dentin-pulp complex, which is a strategic and dynamic barrier in face of injuries suffered by teeth, being caries the most common cause of injury to this complex [1, 2]. The regeneration of these tissues occurs through stimulation and proliferation of mesenchymal progenitor cells, which are attracted to the injury site to differentiate into odontoblast-like cells and produce reparative dentin [6, 7]. Several inflammatory mediators are produced locally to orchestrate the immune response. Among those are the eicosanoids, a class of lipid mediators that are synthesized from arachidonic acid through the action of cyclooxygenases or lipoxygenases to produce prostaglandins and thromboxanes or leukotrienes (LT) and lipoxins, respectively [9, 10]. Because dental pulp inflammation and dentin repair are intrinsically related responses, the aim of this research was to investigate the potential of L TB4 in inducing differentiation of dental pulp stem cells
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