Evaluation of periodontitis-related tooth loss according to the new 2018 classification of periodontitis.

The new 2018 classification of periodontal diseases is reported to be related to tooth loss due to periodontal disease (TLPD) during supportive periodontal therapy (SPT). However, few reports have evaluated this relationship for Asians or have analyzed the association of the new classification and TLPD by distinguishing between active periodontal therapy (APT) and SPT. In this study, we retrospectively applied the new classification to 607 Japanese periodontitis patients and examined the relationship between the new classification and annual TLPD rates per patient during the respective periods. TLPD rates were higher in patients in stage IV and/or grade C during both APT and SPT. TLPD during SPT was not associated with the presence or absence of TLPD during APT. Multivariate analysis revealed that stage IV and grade C as independent variables were significantly associated with the number of instances of TLPD not only during the total treatment period, but also during APT or SPT. Our results suggest that the new classification has a significantly strong association with TLPD during both APT and SPT, and that patients diagnosed with stage IV and/or grade C periodontitis had a higher risk of TLPD during both periods.

Effects of an ascorbic acid-derivative dentifrice in patients with gingivitis: a double-masked, randomized, controlled clinical trial.

BACKGROUND: Reactive oxygen species might be associated with the onset and progression of gingival inflammation. The aim of this study is to investigate the effect of a dentifrice containing L-ascorbic acid 2-phosphate magnesium salt (APM), a long-acting ascorbic acid derivative with antioxidant properties, on gingival inflammation. METHODS: The clinical effects of APM were investigated in a multicenter, randomized, parallel-group, controlled clinical trial comprising 300 individuals with gingivitis. Half of the participants were given an APM-containing dentifrice and half were given a control dentifrice. The primary outcome was the gingival index (GI) at 3 months. Secondary outcomes included gingival redness as an indicator of the degree of local gingival inflammation, gingival bleeding as a measure of the gingivitis severity index, and total antioxidant activity of the saliva. RESULTS: Under the intent-to-treat analysis, GI did not significantly differ between the groups (P = 0.12). However, under the per-protocol analysis, GI was significantly lower in the APM group (P = 0.01) than in the control group. In the APM group, gingival redness was significantly lower, and the difference from the baseline gingivitis severity index was significantly greater (P = 0.04 and P = 0.02, respectively). The total antioxidant activity of the saliva was significantly higher in the APM group (P = 0.03). The incidence of adverse events did not significantly differ between the groups (P > 0.15). CONCLUSION: These findings indicate that the regular application of an APM-containing dentifrice could reduce gingival inflammation.

Nicotine up-regulates IL-8 expression in human gingival epithelial cells following stimulation with IL-1ß or P. gingivalis lipopolysaccharide via nicotinic acetylcholine receptor signalling.

OBJECTIVE: Cigarette smoking is an important risk factor for periodontal disease. The aim of this study is to evaluate the effect of nicotine, a major component of cigarette smoke, on interleukin-8 (IL-8) production and cellular signalling via nicotinic acetylcholine receptors (nAChRs) in human gingival epithelial cells (HGECs). DESIGN: Messenger RNA (mRNA) expression of nAChR subunits in three different HGEC lines (epi 4, Tfx and E6E7) was assessed using reverse transcription-polymerase chain reaction (RT-PCR). HGECs were stimulated by 1×10(-3)M nicotine in the presence or absence of IL-1ß or Porphyromonas gingivalis lipopolysaccharide (LPS). IL-8 production was then examined using real-time PCR and enzyme-linked immunosorbent assay. Nicotine-mediated signalling in the epi 4 cell line was also evaluated by Western blotting. RESULTS: HGECs expressed several nAChR subunits. Nicotine increased the secretion of IL-8 from HGECs that were cultured in the presence of IL-1ß or P. gingivalis LPS and also induced the phosphorylation of extracellular signal-regulated kinase (ERK) in epi 4. Pretreatment with non-selective nAChR antagonist or intracellular calcium chelator reduced the nicotine-induced phosphorylation of ERK. Furthermore, nicotine-induced IL-8 secretion was decreased by pretreatment with non-selective nAChR antagonist, ERK1/2 inhibitor or intracellular calcium chelator. CONCLUSION: These findings indicate that nicotine increases IL-8 production in gingival epithelial cells via ERK phosphorylation following Ca(2+) signalling after nAChR activation.

Fibroblast growth factor-2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction.

Fibroblast growth factor-2 (FGF-2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF-2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF-2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3-kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF-2-stimulated migration of MPDL22 cells. Moreover, in response to FGF-2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)-1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF-2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti-CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF-2. Furthermore, an anti-CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF-2-induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF-2-mediated cell motility of PDL cells, suggesting that FGF-2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production.

Fibroblast growth factor-2 regulates the cell function of human dental pulp cells.

INTRODUCTION: Homeostasis and tissue repair of dentin-pulp complex are attributed to dental pulp tissue and several growth factors. Dental pulp cells play a pivotal role in homeostasis of dentin-pulp complex and tissue responses after tooth injury. Among these cytokines, fibroblast growth factor (FGF)-2 has multifunctional biologic activity and is known as a signaling molecule that induces tissue regeneration. In this study, we examined the effects of FGF-2 on growth, migration, and differentiation of human dental pulp cells (HDPC). METHODS: HDPC were isolated from healthy dental pulp. Cellular response was investigated by [(3)H]-thymidine incorporation into DNA. Cytodifferentiation was examined by alkaline phosphatase (ALPase) assay and cytochemical staining of calcium by using alizarin red. Migratory activity was determined by counting the cells migrating into cleared area that had introduced with silicon block. RESULTS: FGF-2 activated HDPC growth and migration but suppressed ALPase activity and calcified nodule formation. Interestingly, HDPC, which had been pretreated with FGF-2, showed increased ALPase activity and calcified nodule formation when subsequently cultured without FGF-2. These results suggest that FGF-2 potentiates cell growth and accumulation of HDPC that notably did not disturb cytodifferentiation of the cells later. Thus, FGF-2 is a favorable candidate for pulp capping agent. CONCLUSIONS: These results provide new evidence for the possible involvement of FGF-2 not only in homeostasis but also in regeneration of dentin-pulp complex.

Nicotine inhibits mineralization of human dental pulp cells.

Nicotine is a major component of tobacco smoke, and signals via nicotinic acetylcholine receptors (nAChR). However, little is known about the effects of nicotine on human dental pulp cells (HDPCs). In this study, we assessed the effects of nicotine on mineralization in HDPCs. We confirmed messenger RNA expression of nAChR subunits and examined the effects of nicotine on expression of extracellular matrices (ECMs), alkaline phosphatase (ALP) activity, and mineralized nodule formation by HDPCs. Gene expression of nAChR subunits alpha1, alpha2, alpha 4, alpha 5, alpha 6, alpha 7, beta1, beta2, and beta 4 was detected in HDPCs. Interestingly, the messenger RNA expression of dentin matrix acidic phosphoprotein-1, bone sialoprotein, and ALP activity were significantly reduced in nicotine-treated HDPC. In addition, mineralized nodule formation, which was examined by alizarin red staining, was also inhibited in HDPCs by the same treatment. These results indicate that nicotine suppresses the cytodifferentiation and mineralization of HDPCs, possibly via nAChR.

Fibroblast growth factor-2 regulates expression of osteopontin in periodontal ligament cells.

Osteopontin is a protein found in the bone-related matrix and plays multiple regulatory roles in mineralizing and non-mineralizing tissue. In osteogenic cell-lines, the expression of osteopontin increases with the progression of differentiation, but both the expression and function of osteopontin vary with the cell type and its activation state. In this study, we examined the expression of osteopontin by clones established from mouse periodontal ligament, in response to inorganic phosphate and fibroblast growth factor (FGF)-2, which can induce periodontal tissue regeneration. The involvement of inorganic phosphate in the expression of osteopontin during the course of cell differentiation of a clone MPDL22 was confirmed by addition of foscarnet, an inorganic phosphate transport inhibitor. Although FGF-2 decreased the mRNA expression of almost every bone-related protein in MPDL22, FGF-2 upregulated the expression of osteopontin in MPDL22 at both mRNA and protein levels. Interestingly, FGF-2 enhanced the concentration of osteopontin in the culture supernatant of MPDL22, whereas inorganic phosphate did not. The FGF-2-induced osteopontin in the culture supernatant seems to be involved in cell survival activity. An immunohistochemical study showed that the FGF-2-induced osteopontin was mainly present in perinuclear matrices while the inorganic phosphate-induced osteopontin was associated with extracellular matrices in addition to perinuclear matrices. The present results indicated that FGF-2 induces unique expression of osteopontin, which may play a role different from the other bone-related proteins during the process of periodontal tissue regeneration by FGF-2.

Basic fibroblast growth factor regulates expression of heparan sulfate in human periodontal ligament cells.

Heparan sulfate (HS) proteoglycan is a widely distributed biological molecule that mediates a variety of physiological responses in development, cell growth, cell migration, and wound healing. We examined the effects of basic fibroblast growth factor-2 (FGF-2), which is known to modulate extracellular matrix (ECM) production of various cell types, on the production of HS proteoglycan by human periodontal ligament (HPDL) cells. We also examined the effects of FGF-2 on the expression of syndecans, a major family of membrane-bound HS proteoglycans. Treatment of HPDL cells with FGF-2 for 72 h resulted in a pronounced increase in the level of HS in the culture supernatant in a dose-dependent manner. However, reverse transcription-polymerase chain reaction data (RT-PCR) revealed that FGF-2 marginally reduced the gene expression of syndecan-1, -2, and -4, and did not alter the level of syndecan-3 mRNA. Furthermore, FGF-2 did not have an effect on the mRNA expression of enzymes associated with HS biosynthesis. Interestingly, FACS analysis revealed that the syndecan family displayed diverse alterations in response to FGF-2. FGF-2 barely altered the expression of syndecan-1, but decreased the expression of syndecan-2 and -4 on HPDL cells. Moreover, dot blot analysis showed that FGF-2 did not alter the level of syndecan-1 and -2, but enhanced the level of syndecan-4 in culture supernatants of FGF-2-stimulated HPDL cells. These results suggest that the FGF-2-activated increase in the level of HS in conditioned medium may be a result of shedding of syndecan-4 from the HPDL cell surface. Taken together, FGF-2 may differentially regulate the expression of HS proteoglycans in a HS-proteoglycan-subtype-dependent manner. The diversity of the expression patterns of HS proteoglycans may be associated with the FGF-2-induced biological functions of HPDL cells.

Thrombin regulates the function of human blood dendritic cells.

Thrombin is the key enzyme in the coagulation cascade and activates endothelial cells, neutrophils and monocytes via protease-activated receptors (PARs). At the inflammatory site, immune cells have an opportunity to encounter thrombin. However little is known about the effect of thrombin for dendritic cells (DC), which are efficient antigen-presenting cells and play important roles in initiating and regulating immune responses. The present study revealed that thrombin has the ability to stimulate blood DC. Plasmacytoid DC (PDC) and myeloid DC (MDC) isolated from PBMC expressed PAR-1 and released MCP-1, IL-10, and IL-12 after thrombin stimulation. Unlike blood DC, monocyte-derived DC (MoDC), differentiated in vitro did not express PAR-1 and were unresponsive to thrombin. Effects of thrombin on blood DC were significantly diminished by the addition of anti-PAR-1 Ab or hirudin, serine protease inhibitor. Moreover, thrombin induced HLA-DR and CD86 expression on DC and the thrombin-treated DC induced allogenic T cell proliferation. These findings indicate that thrombin plays a role in the regulation of blood DC functions.

Fibroblast growth factor-2 stimulates hyaluronan production by human dental pulp cells.

Hyaluronan (HA), is a high molecular mass extracellular matrix constituting connective tissue and plays a critical role in not only homeostasis but also inflammatory and wound-healing responses. In this study, we investigated the effect of fibroblast growth factor (FGF)-2 on the production of HA by human dental pulp cells (HDPC). An inhibition binding-protein assay showed that FGF-2 increased HA production by HDPC. In addition, expression of mRNA of hyaluronan synthase (HAS) 1 and HAS 2, both of which are related to the production of high molecular mass of HA, but not HAS 3, was enhanced in FGF-2-stimulated HDPC. These results provide new evidence for the involvement of FGF-2 in the regulation of HA production by HDPC possibly through HAS 1 and HAS 2.

Fibroblast growth factor-2 regulates the synthesis of hyaluronan by human periodontal ligament cells.

Basic fibroblast growth factor (FGF-2) can enhance biological potentials of periodontal ligament cells and its topical application induces considerable periodontal tissue regeneration in vivo. In this study, we examined the effect of FGF-2 on the production of hyaluronan (HA), an extracellular matrix playing important roles in homeostasis and inflammatory/wound healing responses, by human periodontal ligament (HPDL) cells. An inhibition binding-protein assay revealed that FGF-2 significantly increased HA production by HPDL cells in a dose dependent manner. Analysis by HPLC revealed that in conditioned medium of FGF-2-treated HPDL cells HA had a higher molecular mass, compared to that of untreated HPDL cells. RT-PCR analysis revealed the enhancement of mRNA expression of hyaluronan synthase (HAS) 1 and HAS 2, both of which contribute to the production of HA with a high molecular mass, but not HAS 3 in the FGF-2-treated HPDL cells. In contrast, three isoforms of hyaluronidase (HYAL) transcript were unchanged in the FGF-2-treated HPDL cells. These results provide new evidence for the possible involvement of FGF-2 in the regulation of HA production and its appreciable roles in not only homeostasis but also regeneration of periodontal tissues.

Human gingival epithelial cells produce chemotactic factors interleukin-8 and monocyte chemoattractant protein-1 after stimulation with Porphyromonas gingivalis via toll-like receptor 2.

BACKGROUND: The mechanism of stimulation of human gingival epithelial cells (HGEC) by Porphyromonas gingivalis (Pg) has not been fully clarified yet. In order to investigate the possible activation of HGEC by Pg through Toll-like receptors (TLRs), we analyzed the production of chemotactic factors and the activated nuclear factor-kappa B (NF-kappaB). METHODS: The mRNA expression of TLRs and the protein expression of TLR2 and TLR4 in HGEC and gingival tissue were assessed using reverse transcription-polymerase chain reaction (RT-PCR) assay and immunohistochemical staining. Primary cultured HGEC (nHGEC) and HGEC transformed by simian virus 40 T antigen (OBA-9) were activated by a sonic extract (SE) of Pg to examine cytokine production and NF-kappaB activation using enzyme-linked immunosorbant assay (ELISA). In addition, Pg mediated activation of NF-kappaB in a TLR2-transfectant was also investigated. RESULTS: RT-PCR results revealed that HGEC expressed mRNA of TLR2, TLR4, TLR5, and TLR9, although the expression profiles of each cell line were slightly different. In addition, immunostaining revealed the prominent expression of TLR2 not only in nHGEC, but also in the gingival epithelium of the tissue specimen. Interestingly, nHGEC and OBA-9 secreted IL-8 and monocyte chemoattractant protein (MCP)-1 upon stimulation with Pg SE more efficiently than LPS and fimbriae of Pg. Furthermore, Pg SE increased the activated NF-kappaB not only in OBA-9, but also in 293T cells transfected with the human TLR2 gene. CONCLUSION: TLR2 participates, at least partly, in the signaling pathway to induce chemokine production in gingival epithelium as a reaction against Pg component(s), probably other than lipopolysaccharide and fimbriae.

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells.

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.