TLR4/7-mediated host-defense responses of gingival epithelial cells.

Gingival epithelial cells (GECs) are physical and immunological barriers against outward pathogens while coping with a plethora of non-pathogenic commensal bacteria. GECs express several members of Toll-like receptors (TLRs) and control subsequent innate immune responses. TLR4 senses lipopolysaccharide (LPS) while TLR7/8 recognizes single-strand RNA (ssRNA) playing important roles against viral infection. However, their distinct roles in GECs have not been fully demonstrated. Here, we analyzed biological responses of GECs to  LPS and CL075, a TLR7/8 agonist. GE1, a mouse gingival epithelial cell line, constitutively express TLR4 and TLR7, but not TLR8, like primary skin keratinocytes. Stimulation of GE1 cells with CL075 induced cytokine, chemokine, and antimicrobial peptide  expressions, the pattern of which is rather different from that with LPS: higher mRNA levels of interferon (IFN) ß, CXCL10, and ß-defensin (BD) 14 (mouse homolog of human BD3); lower levels of tumor necrosis factor (TNF), CCL5, CCL11, CCL20, CXCL2, and CX3CL1. As for the intracellular signal transduction of GE1 cells, CL075 rapidly induced significant AKT phosphorylation but failed to activate IKKα/ß-NFκB pathway, whereas LPS induced marked IKKα/ß-NFκB activation without significant AKT phosphorylation. In contrast, both CL075 and LPS induced rapid IKKα/ß-NFκB activation and AKT phosphorylation in a macrophage cell line. Furthermore, specific inhibition of AKT activity abrogated CL075-induced IFNß, CXCL10, and BD14 mRNA expression in GE1 cells. Thus, TLR4/7 ligands appear to induce rather different host-defense responses of GECs through distinct intracellular signaling mechanisms.

Evaluation of Eruption of Permanent Teeth in Beagle Dog Extraction Sites Filled with Carbonate Apatite.

Autologous bone grafting is the primary method for treating alveolar clefts. However, bone grafting materials are desired as alternatives to autogenous bone to reduce surgical invasiveness. Here, we present an animal study evaluating the effect of carbonate apatite (CA) on the spontaneous eruption of permanent teeth. The bone grafting materials included CA, natural bovine bone (BB), and hydroxyapatite (HA). In 15 8-week-old male beagle dogs, the left mandibular deciduous premolars (DP) two and three were extracted and subsequently filled with CA, BB, and HA. The animals were euthanized after a predetermined number of days, and samples were collected for microcomputed tomography and histological evaluation. Spontaneous eruption of the succeeding permanent teeth (P3 and P4) was observed in the CA group at 14 weeks. Delayed eruption of the succeeding permanent teeth was observed in the BB and HA groups. CA could serve as a viable alternative to autogenous bone for treating alveolar clefts.

Early induction of Hes1 by bone morphogenetic protein 9 plays a regulatory role in osteoblastic differentiation of a mesenchymal stem cell line.

Bone morphogenic protein 9 (BMP9) is one of the most potent inducers of osteogenic differentiation among the 14 BMP members, but its mechanism of action has not been fully demonstrated. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known Notch effector. In this study, we investigated the functional roles of early induction of Hes1 by BMP9 in a mouse mesenchymal stem cell line, ST2. Hes1 mRNA was transiently and periodically induced by BMP9 in ST2, which was inhibited by BMP signal inhibitors but not by Notch inhibitor. Interestingly, Hes1 knockdown in ST2 by siRNA increased the expression of osteogenic differentiation markers such as Sp7 and Ibsp and matrix mineralization in comparison with control siRNA transfected ST2. In contrast, forced expression of Hes1 by using the Tet-On system suppressed the expression of osteogenic markers and matrix mineralization by BMP9. We also found that the early induction of Hes1 by BMP9 suppressed the expression of Alk1, an essential receptor for BMP9. In conclusion, BMP9 rapidly induces the expression of Hes1 via the SMAD pathway in ST2 cells, which plays a negative regulatory role in osteogenic differentiation of mesenchymal stem cells induced by BMP9.

Lipoteichoic Acid and Lipopolysaccharides Are Affected by p38 and Inflammatory Markers and Modulate Their Promoting and Inhibitory Effects on Osteogenic Differentiation.

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are cell wall components of Gram-positive and Gram-negative bacteria, respectively. Notably, oral microflora consists of a variety of bacterial species, and osteomyelitis of the jaw caused by dental infection presents with symptoms of bone resorption and osteosclerosis. However, the effects of LTA and LPS on osteogenic differentiation have not yet been clarified. We examined the effects of LTA and LPS on osteoblasts and found that LTA alone promoted alizarin red staining at low concentrations and inhibited it at high concentrations. Additionally, gene expression of osteogenic markers (ALP, OCN, and OPG) were enhanced at low concentrations of LTA. High concentrations of LPS suppressed calcification potential, and the addition of low concentrations of LTA inhibited calcification suppression, restoring the gene expression levels of suppressed bone differentiation markers (ALP, BSP, and OCN). Moreover, the suppression of p38, a signaling pathway associated with bone differentiation, had opposing effects on gene-level expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), suggesting that mixed LTA and LPS infections have opposite effects on bone differentiation through concentration gradients, involving inflammatory markers (TNF-α and IL-6) and the p38 pathway.