Role of Piezo1 in modulating the RANKL/OPG ratio in mouse osteoblast cells exposed to Porphyromonas gingivalis lipopolysaccharide and mechanical stress.

AIMS: Excessive occlusal force with periodontitis leads to rapid alveolar bone resorption. However, the molecular mechanism by which inflammation and mechanical stress cause bone resorption remains unclear. We examined the role of Piezo1, a mechanosensitive ion channel expressed on osteoblasts, in the changes in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio in mouse MC3T3-E1 osteoblast-like cells under Porphyromonas gingivalis lipopolysaccharide (P.g.-LPS) and mechanical stress. METHODS: To investigate the effect of P.g.-LPS and mechanical stress on the RANKL/OPG ratio and Piezo1 expression, we stimulated MC3T3-E1 cells with P.g.-LPS. After 3 days in culture, shear stress, a form of mechanical stress, was applied to the cells using an orbital shaker. Subsequently, to investigate the role of Piezo1 in the change of RANKL/OPG ratio, we inhibited Piezo1 function by knockdown via Piezo1 siRNA transfection or by adding GsMTx4, a Piezo1 antagonist. RESULTS: The RANKL/OPG ratio significantly increased in MC3T3-E1 cells cultured in a medium containing P.g.-LPS and undergoing mechanical stress compared to cells treated with P.g.-LPS or mechanical stress alone. However, the expression of Piezo1 was not increased by P.g.-LPS and mechanical stress. In addition, phosphorylation of MEK/ERK was induced in the cells under P.g.-LPS and mechanical stress. MC3T3-E1 cells treated with P.g.-LPS and mechanical stress when cocultured with RAW264.7 cells induced their differentiation into osteoclast-like cells. The increased RANKL/OPG ratio was suppressed by either Piezo1 knockdown or the addition of GsMTx4. Furthermore, GsMTx4 inhibited the phosphorylation of MEK/ERK. CONCLUSION: These findings suggest that P.g.-LPS and Piezo1-mediated mechanical stress induce MEK/ERK phosphorylation and increase RANKL expression in osteoblasts. Consequently, this leads to the differentiation of osteoclast precursor cells into osteoclasts.

Implant Site Development by Orthodontic Extrusion and Buccal Root Torque at a Site Showing Severe Gingival Recession with Periodontitis: A Case Report.

This case report describes alveolar ridge augmentation around a hopeless maxillary canine with severe buccal gingival recession and periodontitis using orthodontic extrusion and a 90-degree buccal root torque in preparation for implant placement. After the orthodontic therapy, the palatal surface of the canine root reached the top of the alveolar bone, parallel to the occlusal plane, with newly formed bone and keratinized mucosa. An implant was successfully placed without combined bone augmentation. This technique may be a useful, minimally invasive approach for implant site development where hopeless teeth with severe buccal recession remain in the esthetic areas.

Effects of periodontal treatment on the medical status of patients with type 2 diabetes mellitus: a pilot study.

BACKGROUND: Studies have demonstrated that periodontal disease is associated with the development of systemic complications in patients with type 2 diabetes mellitus (T2DM). The purpose of this pilot study was to investigate which markers among various systemic disease parameters are affected by periodontal treatment in patients with T2DM. METHODS: Twelve patients with T2DM were given oral hygiene instructions and subsequent subgingival scaling and root planing. The periodontal status was recorded, and blood and urine samples were taken to measure various parameters of glucose control and systemic status at baseline and 1 month following the periodontal treatment. Serum concentrations of tumor necrosis factor-α and high-sensitivity C-reactive protein were measured by enzyme-linked immunosorbent assay. RESULTS: After the periodontal treatment, the glycated hemoglobin value was significantly improved. The levels of urinary N-acetyl-ß-D-glucosaminidase and albumin, which are markers of renal dysfunction, also decreased significantly after treatment. Among the parameters measured in serum, the γ-glutamyl transpeptidase level, which is usually interpreted as a marker of liver dysfunction, was significantly reduced. The serum concentrations of tumor necrosis factor-α and high-sensitivity C-reactive protein were also significantly reduced by periodontal treatment. CONCLUSION: Within the limitations of this pilot study, periodontal treatment may be effective not only in improving metabolic control, but also in reducing the risk of diabetic kidney and liver disease in patients with T2DM.

Lysine-specific proteolytic activity responsible for forsythia detaching factor modification.

OBJECTIVES: The objective of the present study was to clarify the lysine-specific proteolytic activity derived from periodontal pathogens responsible for Forsythia detaching factor (FDF) modification. DESIGN: The activity responsible for FDF modification in Tannerella forsythia and Porphyromonas gingivalis were evaluated by colorimetric assay using Ac-Arg-Ala-Lys-p-nitroaniline as a substrate. FDF modification in T. forsythia and P. gingivalis were evaluated by Western blotting using recombinant FDF (rFDF) as a substrate. Furthermore, the activity in GCF of 20 patients with periodontitis and 10 healthy subjects was also evaluated by colorimetric assay. Bacteria in subgingival plaque were detected using polymerase chain reaction. RESULTS: The activity of both bacteria in colorimetric assay were 21.35 unit (P. gingivalis) and 3.61 unit (T. forsythia), respectively. Western blot analysis revealed that P. gingivalis was found to efficiently degrade rFDF and T. forsythia partially cleaved rFDF. The activity in GCF from patients with periodontitis (clinically healthy sites: CH, deep bleeding sites: DB and deep non-bleeding sites: DNB) was significantly higher than those from healthy subjects (healthy sites: H). Among the patients with periodontitis, the activity from CH was significantly lower than those from DB and DNB. T. forsythia was detected in 68.4% of DNB, in 78.4% of DB and in none of CH. P. gingivalis was detected in 63.2% of DNB, in 84.0% of DB and in 10.5% of CH. No bacterium was detected in healthy subjects. CONCLUSION: The lysine-specific proteolytic activity responsible for FDF modification correlates with the presence of major periodontal pathogens.

Modification of forsythia detaching factor by gingival crevicular fluid in periodontitis.

OBJECTIVES: Forsythia detaching factor (FDF) is a virulence factor of Tannerella forsythia detected as a mixture of the 60-kDa form of FDF and the 28-kDa C-terminal fragment (FDFc). The objective of the present study was to clarify the proteolytic activity of gingival crevicular fluid (GCF) from patients with periodontitis and healthy subjects using recombinant FDF (rFDF) as substrate. DESIGN: Eleven patients with periodontitis and 6 healthy subjects were recruited. Modification of rFDF and subsequent production of rFDFc by proteolytic activity of GCF was determined by Western blotting. Proteolytic activity of GCF was evaluated using an Ac-Arg-Ala-Lys-p-nitroaniline substrate. Correlation analysis between two different sets of variables was performed. Variables used in this analysis were proteolytic activity, clinical parameters, relative band density of rFDFc and those of rFDF. RESULTS: Proteolytic activity in GCF was significantly higher in patients with periodontitis than in healthy subjects. Production of rFDFc was determined by treatment of rFDF with GCF from patients with periodontitis and with GCF from healthy subjects. Correlations between clinical parameters and proteolytic activity in GCF were significantly positive. On the other hand, correlations between relative band density of rFDFc or rFDF on Western blot and cleaving activity or clinical parameters were significantly negative. CONCLUSION: The detected extend of GCF-activity generating rFDFc from rFDF and/or even further degrading rFDF correlates with severity of periodontitis.

The significance of podoplanin expression in human inflamed gingiva.

Podoplanin, a transmembrane glycoprotein, has been considered to be expressed specifically by lymphatic endothelial cells. However, recent studies have shown that the protein is expressed in a variety of normal as well as neoplastic tissues, and that its expression might be related to cell migration and invasion. In this study, we examined podoplanin expression in inflamed gingival tissues using an immunohistochemical method. Positive immunoreactivity for podoplanin was found in the cell membrane and cytoplasm of basal cells of oral gingival epithelium when severe inflammatory cell infiltration was present in the connective tissue just under the epithelium. When inflammatory changes were weak or absent, little or no reactivity for podoplanin in the basal cells was observed. Positive reactivity for podoplanin was also detected in basal cell extensions. Surprisingly, strong immunoreactivity for podoplanin was observed in all layers of oral sulcular and junctional epithelia associated with severe inflammatory reaction in the connective tissue. These findings suggest that increased expression of podoplanin in gingival epithelium is related to the progression of chronic periodontitis.

Hypoxia and reoxygenation augment bone-resorbing factor production from human periodontal ligament cells.

BACKGROUND: Oxygen deficiency caused by occlusal trauma and smoking may be associated with bone resorption in periodontitis. In the present study, the effects of hypoxia and reoxygenation on the production of bone-resorbing factors by cultured human periodontal ligament (PDL) cells were examined. METHODS: Human PDL cells were cultured in 1% O(2) (hypoxia), 20% O(2) (normal oxygen tension [normoxia]), or an oxygen concentration that went from 1% to 20% (reoxygenation). The concentrations of bone-resorbing factors, i.e., vascular endothelial growth factor (VEGF), interleukin (IL)-6 and -1beta, tumor necrosis factor-alpha (TNF-alpha), and prostaglandin E(2) (PGE(2)), in the cell culture supernatants were determined by enzyme-linked immunosorbent assay. Expression of the corresponding mRNAs was detected by reverse transcription-polymerase chain reaction. RESULTS: Significantly higher extracellular concentrations of VEGF and IL-6 were detected along with greater corresponding mRNA expression in the hypoxia group compared to the normoxia group. The protein production and mRNA expression of IL-1beta were observed only in the hypoxia group. Neither TNF-alpha nor PGE(2) was detectable in samples from either group, whereas cyclooxygenase-2 mRNA was detected. However, PGE(2) was detected after reoxygenation. Furthermore, VEGF and IL-6 and -1beta production also tended to increase in extracellular concentration and mRNA level after reoxygenation. CONCLUSION: Hypoxia and reoxygenation may stimulate the PDL to produce VEGF, IL-6 and -1beta, and PGE2, which could result in the resorption of alveolar bone in periodontitis.

High prevalence of Helicobacter pylori detected by PCR in the oral cavities of periodontitis patients.

BACKGROUND: Helicobacter pylori has been associated with the development of peptic ulcers and gastric cancer. Although it may be transmitted through the oral cavity, it is unknown whether the oral cavity acts as a permanent reservoir for this bacterium. The purpose of this study was to use nested polymerase chain reaction (PCR) to clarify whether the oral cavity acts as a reservoir for H. pylori. METHODS: The existence of H. pylori in the oral cavity was determined by nested PCR in 57 subjects and by culture method in 18 subjects. The presence of periodontopathic bacteria was also determined by 16S rRNA-based PCR method. RESULTS: Although H. pylori was rarely detected in the oral cavity by culture technique, it was frequently detected (35.1%) by nested PCR in the oral cavity, especially among periodontitis patients who had the bacterium in the gastrointestinal tract (46.4%). Among the subjects who harbored H. pylori in the stomach or duodenum, 41.2% of patients with periodontal pockets > or = 4 mm and 9.1% of subjects without pockets showed H. pylori in dental plaque, although a statistically significant difference was not observed. One patient who had periodontal pockets retained H. pylori in the oral cavity even after eradication of the bacterium from the stomach and duodenum. Most (8/10) of the patients who had H. pylori in dental plaque harbored Bacteroides forsythus in their oral cavities. CONCLUSION: Close attention should be given to periodontitis patients who harbor H. pylori in the oral cavity.

Elevated kallikrein activity in plasma from stable liver transplant recipients.

Extensive in vitro conversion of complement components C3 and C4 has been observed in EDTA plasma obtained from a number of stable orthotopic liver transplant recipients (LTR) [Clin. Chem. 45 (1999) 1190]. Consequently, we designed a chromogenic substrate (Ac-Ala-Gly-Leu-Thr-Arg-p-nitroanilide, AGLTR-pNA), based on the C1s cleavage site in complement component C4, in an attempt to identify the plasma proteinase(s) that cleaves C4 in vitro. Average peptidase activity in EDTA plasma obtained from stable LTR (n = 16) was significantly higher (P<0.01) than that in plasma from healthy non-transplant donors (n = 16). This peptidase activity was also detected using commercial substrates designed for specific coagulation proteinases. The plasma proteinase was not inhibited by hirudin, a thrombin inhibitor, but was inhibited by the plasma kallikrein inhibitor D-Phe-Phe-Arg-chloromethylketone, which fails to inhibit C1s. We concluded that the peptidase detected inLTR plasma, using chromogenic substrates including AGLTR-pNA, was plasma kallikrein. Western blot analysis confirmed the presence of kallikrein-alpha-2-macroglobulin complexes (alpha2M) in LTR plasmas. We also demonstrated that kallikrein was not the proteinase responsible for the in vitro cleavage of C4. Elevation of the plasma peptidase activity correlated significantly with recurrent hepatitis C virus (HCV) infection in these liver recipients with a P value <0.02. Significant correlation was not observed between complement activation (i.e. the C4a levels) and recurrent HCV infection (P>0.15); however, C4a levels did correlate with rejection (P<0.02). These results suggest that elevation in plasma peptidase activity and activation of complement do signal different pathological events in LTR, events that appear related to HCV-induced infection and immune tissue injury, respectively.