Calcium phosphate particles induce interleukin-8 expression in a human gingival epithelial cell line via the nuclear factor-κB signaling pathway.

BACKGROUND: Dental calculus is calcified plaque composed primarily of calcium phosphate mineral salts, and there is a clear association between the presence of calculus and the initiation/progression of periodontitis. However, it is still inconclusive whether dental calculus can be a direct causative factor. The authors examined the effect of nano/microsized calcium phosphate particles, which may be generated in the process of early precipitation and/or dissolution of calcium phosphate mineral, on the expression of interleukin (IL)-8 in human gingival epithelial cells. METHODS: Primary human gingival epithelial cells and/or a human gingival carcinoma cell line (Ca9-22) were stimulated with calcium phosphate particles. Gene and protein levels were assessed by real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay, respectively. The activity of nuclear factor (NF)-κB signaling was measured by an immunofluorescence assay to evaluate NF-κB p65 nuclear translocation. RESULTS: The results show that nano/microsized particles stimulate IL-8 expression in human gingival epithelial cells at gene and protein levels. The activity to induce IL-8 expression depends on the particle size: particles with a diameter of 200 nm are more effective than those of 40-nm and 5-µm diameters. Calcium phosphate particles (diameter 200 nm) stimulated NF-κB activity. Pretreatment with BMS-345541, an NF-κB signaling inhibitor, inhibited the particle-mediated IL-8 gene induction, suggesting a requirement for the NF-κB signaling pathway. CONCLUSION: These findings suggest that calcium phosphate particles, which may be related to calculus development, may act as a direct causative factor in the pathogenesis of gingival epithelium.

Epithelial-mesenchymal interactions induce enamel matrix proteins and proteases in the epithelial cells of the rests of Malassez in vitro.

Epithelial-mesenchymal interactions influence morphogenesis and cell differentiation in periodontal tissue regeneration. The current study examined the expression of amelogenin, ameloblastin, matrix metallopeptidase-20 (MMP-20), and kallikrein-4 (KLK-4) and their effects on the interactions between the epithelial cells of Malassez and periodontal ligament fibroblasts. Explants of human periodontal ligament tissues produced outgrowths containing both the epithelial cells of Malassez and periodontal ligament fibroblasts after incubation in a modified serum-free medium. Both the epithelial cells and fibroblasts were co-cultured in the same dish. The distribution and expression of all four factors were evaluated using immunohistochemistry, in-situ hybridization and RT-PCR analysis. The epithelial cells of Malassez were cultured separately and were used as the control. Immunohistochemical analysis revealed weak expression of amelogenin, ameloblastin, MMP-20 and KLK-4 in epithelial cells of Malassez co-cultured with periodontal ligament fibroblasts. in-situ hybridization and RT-PCR confirmed significant mRNA expression of these factors in co-cultured cells compared with control cells. MMP20 mRNA was not expressed in control cells. These results suggest that the epithelial-mesenchymal interactions promote differentiation of human epithelial cells of Malassez and that the induction of enamel matrix proteases facilitates the degradation of enamel matrix proteins.

Expression of type IV collagen and laminin at the interface between epithelial cells and fibroblasts from human periodontal ligament.

The present study was undertaken to examine whether synthesis of type IV collagen and laminin around the epithelial rests of Malassez (ERM) requires direct contact between cells from ERM and periodontal ligament fibroblasts. Human periodontal ligament (HPDL) explants produced outgrowths containing both ERM cells and fibroblasts when cultured in a modified serum-free medium. The interface between ERM cells and fibroblasts was examined using phase-contrast microscopy (PCM) and scanning electron microscopy (SEM). Expression of type IV collagen and laminin was studied by immunohistochemistry and in situ hybridization. It was observed that ERM cells grew underneath fibroblasts or attached to them. At the interface, type IV collagen and laminin and their respective mRNAs were abundant in both ERM cells and fibroblasts, while these proteins and mRNAs showed little if any staining in cells further away from the interface. Hence, these findings indicate that synthesis of type IV collagen and laminin is induced by direct interaction between ERM cells and periodontal ligament fibroblasts.

The involvement of platelet-derived growth factor receptors and insulin-like growth factor-I receptors signaling during mineralized nodule formation by human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Periodontal ligament cells are regarded to have the capacity to differentiate into cementoblasts or osteoblasts, and are capable of forming a mineralized nodule in vitro. However, the precise mechanisms are unclear. Here we evaluated the possible involvement of growth factor receptors, such as the platelet-derived growth factor receptor (PDGFR), insulin-like growth factor-I receptor (IGF-IR), and epidermal growth factor receptor (EGFR) on periodontal ligament cells and their ligands during periodontal ligament cells differentiation in vitro. METHODS: Human periodontal ligament cells were differentiated via culturing in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate for mineralized nodule formation, characterized by von Kossa staining. Expressions of receptors and their ligands were analyzed by flow cytometry/reverse transcription-polymerase chain reaction. RESULTS: During the differentiation, PDGFR-alpha was held at a lower level compared with the control. PDGFR-beta, however, was maintained at a slightly higher level that was reversed to the control level when mineralized nodules formed. In contrast, IGF-IR and EGFR were not substantially different from the control. The mineralized nodule formation was strongly inhibited by a PDGFR kinase blocker (AG1295 and AG1296), partially inhibited by an IGF-IR kinase blocker (I-Ome-AG538 and AG1024), and not inhibited by an EGFR kinase blocker (AG99). PDGF-A, PDGF-C, PDGF-D, IGF-I, and IGF-II, but not PDGF-B, were expressed on the control as well as dexamethasone/ascorbic acid-treated periodontal ligament cells during mineralized nodule formation; however, the pattern of their expressions was quite different. CONCLUSION: These findings suggest that a pathway of PDGFs/PDGFR and IGFs/IGF-IR on periodontal ligament cells are involved during mineralized nodule formation, and that PDGFs and IGFs expressed by periodontal ligament cells may contribute to the formation.