Effect of lipopolysaccharide stimulation on stem cell-associated marker-expressing cells.

Abstract

To examine the effect of inflammatory stimuli on the proliferation/migration of dental pulp stem cells by assessing the responses of stem cell-associated marker-expressing cells in rat incisors to lipopolysaccharide (LPS) stimulation invivo. The crowns of rat incisors were removed, and the coronal pulp chamber was instrumented. After haemostasis, an absorbent point soaked in LPS was inserted into the cavity, which was then sealed. At 3, 12, and 48h after LPS application, pulp tissues were subjected to double-immunoperoxidase labelling using two of the antibodies against microtubule-associated protein 1B (MAP1B), CD146 and STRO-1. For gene expression analysis, total RNA was extracted, and mRNA expression levels of stem cell factor (SCF), stromal-derived factor 1 (SDF-1), CD146 and MAP1B were analysed with real-time polymerase chain reaction. SCF and SDF-1 protein levels were also assessed by Western blot. Statistical analysis was performed by Kruskal-Wallis nonparametric analysis of variance, followed by Mann-Whitney U-tests with Bonferroni correction. The density of MAP1B+ CD146+ cells and STRO-1+ CD146+ cells in LPS-stimulated pulp tissue increased significantly at 3h and exhibited a four- to sixfold increase at 48h as compared with the density observed in normal pulp tissue (P<0.05). The expression of CD146 mRNA in LPS-stimulated pulp showed significant upregulation at 3h as compared with that observed in normal pulp tissue (P<0.05). MAP1B, SCF and SDF-1 mRNA levels also showed significant upregulation at 3 and 72h (P<0.05), and Western blot analysis revealed increases in SCF and SDF-1 following LPS stimulation. LPS-stimulated pulp tissue exhibited upregulation of stem cell differentiation/migration markers and showed increases in the number of MAP1B+ CD146+ and STRO-1+ CD146 stem-like cells.